Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury
Identifieur interne : 001F16 ( Istex/Corpus ); précédent : 001F15; suivant : 001F17Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury
Auteurs : Masaru Shinyashiki ; Yoshito Kumagai ; Hiromi Nakajima ; Jun Nagafune ; Shino Homma-Takeda ; Masaru Sagai ; Nobuhiro ShimojoSource :
- Brain Research [ 0006-8993 ] ; 1998.
English descriptors
- KwdEn :
- Teeft :
- Brain research, Cerebellar, Cerebellum, Cerebrum, Continuous exposure, Covalent modification, Dismutase, Enzyme, Enzyme activity, Enzyme loss, Enzyme preparation, First injection, Gene expression, Immunoblot analysis, Incubation mixture, Inos, Intracellular, Isozymes, Kgy1 dayy1, Macrophage, Macrophage inos, Manganese superoxide dismutase, Mercurials, Mercuric, Mercuric chloride, Mercuric compounds, Mercury compounds, Methylmercuric chloride, Methylmercury, Mgy1, Miny1, Mrna, Neuron, Nitric, Nitric oxide, Nitric oxide synthase, Nmol, Nmol miny1 mgy1, Nnos, Nnos activity, Nnos protein, Oxide, Pharmacol, Primer, Protein levels, Purkinje cells, Reaction mixture, Shinyashiki, Slight modification, Sulfhydryl, Sulfhydryl compounds, Supernatant, Superoxide, Synthase, Thiol, Toxicol, Tsukuba, Western blot analysis.
Abstract
Abstract: Alterations in mRNA level, protein content and enzyme activity for nitric oxide synthase (NOS) in the cerebrum and cerebellum during a continuous exposure of neurotoxic metal, methylmercury, were examined in Wistar rats. Subcutaneous (s.c.) administration of methylmercuric chloride (MMC, 10 mg kg−1 day−1, 8 days) resulted in significant increases with time of NOS activities in the cerebrum (1.6–1.9-fold, 5–8 days) and cerebellum (1.4-fold, 8 days). RT-PCR and immunoblot analyses indicated that the increase in the enzyme activity caused by this metal appears to be due to increase in protein levels of neuronal NOS (nNOS), but not inducible NOS (iNOS) because little appreciable mRNA and protein for iNOS were seen during MMC exposure. The direct effect of mercuric compounds on nNOS activity in vitro was evaluated using 20,000×g supernatant from rat cerebellum homogenate. In contrast to the in vivo observation, inorganic-, alkyl-, and aryl-mercuric compound showed potent inhibition of nNOS activity with IC50 values of 11–43 μM, whereas dimethylmercury (DMM) was without effect on the enzyme activity. Further experiments indicated that the inhibition of nNOS by organomercurial occurred via thiol modification.
Url:
DOI: 10.1016/S0006-8993(98)00400-4
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ISTEX:DF9D5B1F6BF3A2ECD438635FB4C0C901141D6D12Le document en format XML
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uniqKey="Nakajima H" first="Hiromi" last="Nakajima">Hiromi Nakajima</name><affiliation><mods:affiliation>Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</mods:affiliation></affiliation></author><author><name sortKey="Nagafune, Jun" sort="Nagafune, Jun" uniqKey="Nagafune J" first="Jun" last="Nagafune">Jun Nagafune</name><affiliation><mods:affiliation>Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</mods:affiliation></affiliation></author><author><name sortKey="Homma Takeda, Shino" sort="Homma Takeda, Shino" uniqKey="Homma Takeda S" first="Shino" last="Homma-Takeda">Shino Homma-Takeda</name><affiliation><mods:affiliation>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</mods:affiliation></affiliation></author><author><name sortKey="Sagai, Masaru" sort="Sagai, Masaru" 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ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Alterations in mRNA level, protein content and enzyme activity for nitric oxide synthase (NOS) in the cerebrum and cerebellum during a continuous exposure of neurotoxic metal, methylmercury, were examined in Wistar rats. Subcutaneous (s.c.) administration of methylmercuric chloride (MMC, 10 mg kg−1 day−1, 8 days) resulted in significant increases with time of NOS activities in the cerebrum (1.6–1.9-fold, 5–8 days) and cerebellum (1.4-fold, 8 days). RT-PCR and immunoblot analyses indicated that the increase in the enzyme activity caused by this metal appears to be due to increase in protein levels of neuronal NOS (nNOS), but not inducible NOS (iNOS) because little appreciable mRNA and protein for iNOS were seen during MMC exposure. The direct effect of mercuric compounds on nNOS activity in vitro was evaluated using 20,000×g supernatant from rat cerebellum homogenate. In contrast to the in vivo observation, inorganic-, alkyl-, and aryl-mercuric compound showed potent inhibition of nNOS activity with IC50 values of 11–43 μM, whereas dimethylmercury (DMM) was without effect on the enzyme activity. Further experiments indicated that the inhibition of nNOS by organomercurial occurred via thiol modification.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>nnos</json:string><json:string>nitric</json:string><json:string>inos</json:string><json:string>methylmercury</json:string><json:string>nnos activity</json:string><json:string>cerebrum</json:string><json:string>enzyme activity</json:string><json:string>mercuric</json:string><json:string>synthase</json:string><json:string>cerebellum</json:string><json:string>nmol</json:string><json:string>miny1</json:string><json:string>mgy1</json:string><json:string>shinyashiki</json:string><json:string>mercuric compounds</json:string><json:string>thiol</json:string><json:string>pharmacol</json:string><json:string>sulfhydryl</json:string><json:string>macrophage</json:string><json:string>mercurials</json:string><json:string>brain 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Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</json:string></affiliations></json:item><json:item><name>Hiromi Nakajima</name><affiliations><json:string>Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</json:string></affiliations></json:item><json:item><name>Jun Nagafune</name><affiliations><json:string>Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</json:string></affiliations></json:item><json:item><name>Shino Homma-Takeda</name><affiliations><json:string>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</json:string></affiliations></json:item><json:item><name>Masaru Sagai</name><affiliations><json:string>National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305, Japan</json:string></affiliations></json:item><json:item><name>Nobuhiro Shimojo</name><affiliations><json:string>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Nitric oxide synthase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Enzyme activity</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Methylmercuric chloride</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Mercuric chloride</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Thiol compounds</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Inhibition</value></json:item></subject><articleId><json:string>14633</json:string></articleId><arkIstex>ark:/67375/6H6-N6Z0091G-T</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Alterations in mRNA level, protein content and enzyme activity for nitric oxide synthase (NOS) in the cerebrum and cerebellum during a continuous exposure of neurotoxic metal, methylmercury, were examined in Wistar rats. Subcutaneous (s.c.) administration of methylmercuric chloride (MMC, 10 mg kg−1 day−1, 8 days) resulted in significant increases with time of NOS activities in the cerebrum (1.6–1.9-fold, 5–8 days) and cerebellum (1.4-fold, 8 days). RT-PCR and immunoblot analyses indicated that the increase in the enzyme activity caused by this metal appears to be due to increase in protein levels of neuronal NOS (nNOS), but not inducible NOS (iNOS) because little appreciable mRNA and protein for iNOS were seen during MMC exposure. The direct effect of mercuric compounds on nNOS activity in vitro was evaluated using 20,000×g supernatant from rat cerebellum homogenate. In contrast to the in vivo observation, inorganic-, alkyl-, and aryl-mercuric compound showed potent inhibition of nNOS activity with IC50 values of 11–43 μM, whereas dimethylmercury (DMM) was without effect on the enzyme activity. Further experiments indicated that the inhibition of nNOS by organomercurial occurred via thiol modification.</abstract><qualityIndicators><score>9.172</score><pdfWordCount>5864</pdfWordCount><pdfCharCount>35456</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>9</pdfPageCount><pdfPageSize>595 x 738 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>181</abstractWordCount><abstractCharCount>1231</abstractCharCount><keywordCount>6</keywordCount></qualityIndicators><title>Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury</title><pmid><json:string>9666107</json:string></pmid><pii><json:string>S0006-8993(98)00400-4</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Brain Research</title><language><json:string>unknown</json:string></language><publicationDate>1998</publicationDate><issn><json:string>0006-8993</json:string></issn><pii><json:string>S0006-8993(00)X0167-9</json:string></pii><volume>798</volume><issue>1–2</issue><pages><first>147</first><last>155</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1-1-1</json:string><json:string>1998</json:string></date><geogName></geogName><orgName><json:string>Research Products</json:string><json:string>Biomedical Technologies</json:string><json:string>Ministry of Education, Science and Culture, Japan</json:string><json:string>Merck Sharp and Dohme Laboratories</json:string><json:string>Japan Accepted</json:string><json:string>Meijo University</json:string><json:string>Bio-Rad Laboratories</json:string><json:string>Institute of Community Medicine</json:string><json:string>National Institute</json:string><json:string>Scientific Research Grantin-Aid</json:string></orgName><orgName_funder><json:string>Scientific Research Grantin-Aid</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Research</json:string><json:string>Brain Research</json:string><json:string>Ždata</json:string><json:string>Western</json:string><json:string>A. Electrophoresis</json:string><json:string>Y. Komori</json:string></persName><placeName><json:string>Bradford</json:string><json:string>Toyama</json:string><json:string>Nagoya</json:string><json:string>Japan</json:string><json:string>Tokyo</json:string><json:string>Osaka</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Kerper et al.</json:string><json:string>Komori et al.</json:string><json:string>Jacobs et al.</json:string><json:string>M. Shinyashiki et al.</json:string><json:string>Schmidt et al.</json:string><json:string>Lynas et al.</json:string><json:string>Himi et al.</json:string><json:string>Klein et al.</json:string><json:string>Towbin et al.</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-N6Z0091G-T</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - neurosciences</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - clinical medicine</json:string><json:string>3 - neurology & neurosurgery</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Developmental Biology</json:string><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Clinical Neurology</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Molecular Biology</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Neuroscience</json:string><json:string>3 - General Neuroscience</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences medicales</json:string></inist></categories><publicationDate>1998</publicationDate><copyrightDate>1998</copyrightDate><doi><json:string>10.1016/S0006-8993(98)00400-4</json:string></doi><id>DF9D5B1F6BF3A2ECD438635FB4C0C901141D6D12</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-N6Z0091G-T/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-N6Z0091G-T/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-N6Z0091G-T/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1998 Elsevier Science B.V.</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1998</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Section title: Research report</note><note type="content">Fig. 1: Effects of continuous injection with MMC on NOS activities in the cerebrum (A) and cerebellum (B) of rat. Rats were injected with MMC (10 mg kg−1 day−1) for 8 days. Zero day represents control group. Data is the mean±S.D. of five animals. Differences compared to control groups were analyzed by One-way ANOVA: *, P<0.05; **, P<0.01.</note><note type="content">Fig. 2: RT-PCR analysis of mRNA levels of NOS isozymes in the cerebrum and cerebellum during exposure of rats to MMC. (A) RT-PCR products for nNOS, iNOS and β-actin over a 14-day time course; (B) quantification of the RT-PCR products from the experiments illustrated in A (nNOS, closed square; β-actin, open square). Extraction of total RNA and RT-PCR were performed under the conditions described in Section 2. C and P indicate control and paralysis, respectively. Each point is the average of two determinations. The alteration shown in A is a representative of data from four other animals.</note><note type="content">Fig. 3: Immunoblot analysis of protein levels of NOS isozymes in the cerebrum and cerebellum during exposure of rats to MMC. (A) protein that react with specific antibodies against nNOS and iNOS over a 14-day time course; (B) quantification of the immunoreactive proteins from the experiments illustrated in A. Electrophoresis and Western blot analysis were performed under conditions described in Section 2. C and P indicate control and paralysis, respectively. Each point is the average of two determinations. Purified mouse macrophage iNOS was used as the standard. The alteration shown in A is a representative of data from four other animals.</note><note type="content">Fig. 4: Effects of mercury derivatives on NOS activity in rat cerebellum 20,000×g supernatant. Rat cerebellum 20,000×g supernatants were incubated with a different concentration of MC (closed square), MMC (closed circle), EMC (open circle), PMC (open square) or DMM (closed triangle). Reaction mixtures (0.1 ml) containing enzyme preparation (70–116 μg of protein), 40 mM HEPES (pH 7.6) in the absence and presence of mercury compounds were preincubated at 37°C for 5 min. Mercury compounds were dissolved with different solvents as follows: MC, MMC and EMC, water; PMC, dimethyl sulfoxide (final conc., 2%); DMM, ethanol (final conc., 2%). Data indicate relative inhibition to control NOS activities with water, dimethyl sulfoxide and ethanol as solvent. Each data is the mean of duplicate experiments.</note><note type="content">Fig. 5: Elution patterns of rat cerebellum nNOS from Affi-Gel 501 column. The 20,000×g supernatant of treated rat cerebellum, following Econo-Pac 10 DG column chromatography (2.0 mg of protein) was applied to the Affi-Gel 501 column (2.0×1.0 cm, i.d.) and bound proteins were successively eluted by 5 vol. of 0.5 M NaCl (a), followed by 0.05% Tween 20 (b), 10 mM histidine (c), and 10 mM 2-ME (d) in the 50 mM Tris–HCl (pH 7.4)–0.1 mM EDTA–0.1 mM EGTA–1 μM pepstatin A–2 μM leupeptin–1 mM PMSF. Each fraction (2.1 ml) collected was employed to determine enzyme activity and to conduct immunoblot analysis. Solid lines and closed squares indicate absorbance at 280 nm and NOS activity, respectively. The eluates were electrophoresed by SDS-PAGE, followed by Western blotting by the method describing Section 2. The bands in the figure indicate; Lane 1, rat nNOS (43 ng); lane 2, macrophage iNOS (42 ng); lane 3, fraction 25 (4.1 μg).</note><note type="content">Table 1: Changes in total mercury concentration in the blood, cerebrum and cerebellum from rat subcutaneously injected with MMC (10 mg kg−1 day−1) for 8 days</note><note type="content">Table 2: Effects of pre- and post-treatment with SH compounds on the inhibition of nNOS activity caused by MC and MMC</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury</title><author xml:id="author-0000"><persName><forename type="first">Masaru</forename><surname>Shinyashiki</surname></persName><affiliation>Graduate School Doctoral Program in Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Yoshito</forename><surname>Kumagai</surname></persName><note type="correspondence"><p>Corresponding author. Fax: +81-0298-53-3039.</p></note><affiliation>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Hiromi</forename><surname>Nakajima</surname></persName><affiliation>Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Jun</forename><surname>Nagafune</surname></persName><affiliation>Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Shino</forename><surname>Homma-Takeda</surname></persName><affiliation>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Masaru</forename><surname>Sagai</surname></persName><affiliation>National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305, Japan</affiliation></author><author xml:id="author-0006"><persName><forename type="first">Nobuhiro</forename><surname>Shimojo</surname></persName><affiliation>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation></author><idno type="istex">DF9D5B1F6BF3A2ECD438635FB4C0C901141D6D12</idno><idno type="ark">ark:/67375/6H6-N6Z0091G-T</idno><idno type="DOI">10.1016/S0006-8993(98)00400-4</idno><idno type="PII">S0006-8993(98)00400-4</idno><idno type="article-id">14633</idno></analytic><monogr><title level="j">Brain Research</title><title level="j" type="abbrev">BRES</title><idno type="pISSN">0006-8993</idno><idno type="PII">S0006-8993(00)X0167-9</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998"></date><biblScope unit="volume">798</biblScope><biblScope unit="issue">1–2</biblScope><biblScope unit="page" from="147">147</biblScope><biblScope unit="page" to="155">155</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1998</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Alterations in mRNA level, protein content and enzyme activity for nitric oxide synthase (NOS) in the cerebrum and cerebellum during a continuous exposure of neurotoxic metal, methylmercury, were examined in Wistar rats. Subcutaneous (s.c.) administration of methylmercuric chloride (MMC, 10 mg kg−1 day−1, 8 days) resulted in significant increases with time of NOS activities in the cerebrum (1.6–1.9-fold, 5–8 days) and cerebellum (1.4-fold, 8 days). RT-PCR and immunoblot analyses indicated that the increase in the enzyme activity caused by this metal appears to be due to increase in protein levels of neuronal NOS (nNOS), but not inducible NOS (iNOS) because little appreciable mRNA and protein for iNOS were seen during MMC exposure. The direct effect of mercuric compounds on nNOS activity in vitro was evaluated using 20,000×g supernatant from rat cerebellum homogenate. In contrast to the in vivo observation, inorganic-, alkyl-, and aryl-mercuric compound showed potent inhibition of nNOS activity with IC50 values of 11–43 μM, whereas dimethylmercury (DMM) was without effect on the enzyme activity. Further experiments indicated that the inhibition of nNOS by organomercurial occurred via thiol modification.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Nitric oxide synthase</term></item><item><term>Enzyme activity</term></item><item><term>Methylmercuric chloride</term></item><item><term>Mercuric chloride</term></item><item><term>Thiol compounds</term></item><item><term>Inhibition</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1998">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-N6Z0091G-T/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>BRES</jid><aid>14633</aid><ce:pii>S0006-8993(98)00400-4</ce:pii><ce:doi>10.1016/S0006-8993(98)00400-4</ce:doi><ce:copyright year="1998" type="full-transfer">Elsevier Science B.V.</ce:copyright></item-info><head><ce:dochead><ce:textfn>Research report</ce:textfn></ce:dochead><ce:title>Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury</ce:title><ce:author-group><ce:author><ce:given-name>Masaru</ce:given-name><ce:surname>Shinyashiki</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref></ce:author><ce:author><ce:given-name>Yoshito</ce:given-name><ce:surname>Kumagai</ce:surname><ce:cross-ref refid="AFF2">b</ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref></ce:author><ce:author><ce:given-name>Hiromi</ce:given-name><ce:surname>Nakajima</ce:surname><ce:cross-ref refid="AFF3">c</ce:cross-ref></ce:author><ce:author><ce:given-name>Jun</ce:given-name><ce:surname>Nagafune</ce:surname><ce:cross-ref refid="AFF3">c</ce:cross-ref></ce:author><ce:author><ce:given-name>Shino</ce:given-name><ce:surname>Homma-Takeda</ce:surname><ce:cross-ref refid="AFF2">b</ce:cross-ref></ce:author><ce:author><ce:given-name>Masaru</ce:given-name><ce:surname>Sagai</ce:surname><ce:cross-ref refid="AFF4">d</ce:cross-ref></ce:author><ce:author><ce:given-name>Nobuhiro</ce:given-name><ce:surname>Shimojo</ce:surname><ce:cross-ref refid="AFF2">b</ce:cross-ref></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Graduate School Doctoral Program in Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</ce:textfn></ce:affiliation><ce:affiliation id="AFF3"><ce:label>c</ce:label><ce:textfn>Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</ce:textfn></ce:affiliation><ce:affiliation id="AFF4"><ce:label>d</ce:label><ce:textfn>National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305, Japan</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Fax: +81-0298-53-3039.</ce:text></ce:correspondence></ce:author-group><ce:date-accepted day="7" month="4" year="1998"></ce:date-accepted><ce:abstract class="author"><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para view="all" id="simple-para.0080">Alterations in mRNA level, protein content and enzyme activity for nitric oxide synthase (NOS) in the cerebrum and cerebellum during a continuous exposure of neurotoxic metal, methylmercury, were examined in Wistar rats. Subcutaneous (s.c.) administration of methylmercuric chloride (MMC, 10 mg kg<ce:sup loc="post">−1</ce:sup> day<ce:sup loc="post">−1</ce:sup>, 8 days) resulted in significant increases with time of NOS activities in the cerebrum (1.6–1.9-fold, 5–8 days) and cerebellum (1.4-fold, 8 days). RT-PCR and immunoblot analyses indicated that the increase in the enzyme activity caused by this metal appears to be due to increase in protein levels of neuronal NOS (nNOS), but not inducible NOS (iNOS) because little appreciable mRNA and protein for iNOS were seen during MMC exposure. The direct effect of mercuric compounds on nNOS activity in vitro was evaluated using 20,000×<ce:italic>g</ce:italic> supernatant from rat cerebellum homogenate. In contrast to the in vivo observation, inorganic-, alkyl-, and aryl-mercuric compound showed potent inhibition of nNOS activity with IC<ce:inf loc="post">50</ce:inf> values of 11–43 <ce:italic>μ</ce:italic>M, whereas dimethylmercury (DMM) was without effect on the enzyme activity. Further experiments indicated that the inhibition of nNOS by organomercurial occurred via thiol modification.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Nitric oxide synthase</ce:text></ce:keyword><ce:keyword><ce:text>Enzyme activity</ce:text></ce:keyword><ce:keyword><ce:text>Methylmercuric chloride</ce:text></ce:keyword><ce:keyword><ce:text>Mercuric chloride</ce:text></ce:keyword><ce:keyword><ce:text>Thiol compounds</ce:text></ce:keyword><ce:keyword><ce:text>Inhibition</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury</title></titleInfo><name type="personal"><namePart type="given">Masaru</namePart><namePart type="family">Shinyashiki</namePart><affiliation>Graduate School Doctoral Program in Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Yoshito</namePart><namePart type="family">Kumagai</namePart><affiliation>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation><description>Corresponding author. Fax: +81-0298-53-3039.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Hiromi</namePart><namePart type="family">Nakajima</namePart><affiliation>Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Jun</namePart><namePart type="family">Nagafune</namePart><affiliation>Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Shino</namePart><namePart type="family">Homma-Takeda</namePart><affiliation>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Masaru</namePart><namePart type="family">Sagai</namePart><affiliation>National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Nobuhiro</namePart><namePart type="family">Shimojo</namePart><affiliation>Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1998</dateIssued><copyrightDate encoding="w3cdtf">1998</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Alterations in mRNA level, protein content and enzyme activity for nitric oxide synthase (NOS) in the cerebrum and cerebellum during a continuous exposure of neurotoxic metal, methylmercury, were examined in Wistar rats. Subcutaneous (s.c.) administration of methylmercuric chloride (MMC, 10 mg kg−1 day−1, 8 days) resulted in significant increases with time of NOS activities in the cerebrum (1.6–1.9-fold, 5–8 days) and cerebellum (1.4-fold, 8 days). RT-PCR and immunoblot analyses indicated that the increase in the enzyme activity caused by this metal appears to be due to increase in protein levels of neuronal NOS (nNOS), but not inducible NOS (iNOS) because little appreciable mRNA and protein for iNOS were seen during MMC exposure. The direct effect of mercuric compounds on nNOS activity in vitro was evaluated using 20,000×g supernatant from rat cerebellum homogenate. In contrast to the in vivo observation, inorganic-, alkyl-, and aryl-mercuric compound showed potent inhibition of nNOS activity with IC50 values of 11–43 μM, whereas dimethylmercury (DMM) was without effect on the enzyme activity. Further experiments indicated that the inhibition of nNOS by organomercurial occurred via thiol modification.</abstract><note type="content">Section title: Research report</note><note type="content">Fig. 1: Effects of continuous injection with MMC on NOS activities in the cerebrum (A) and cerebellum (B) of rat. Rats were injected with MMC (10 mg kg−1 day−1) for 8 days. Zero day represents control group. Data is the mean±S.D. of five animals. Differences compared to control groups were analyzed by One-way ANOVA: *, P<0.05; **, P<0.01.</note><note type="content">Fig. 2: RT-PCR analysis of mRNA levels of NOS isozymes in the cerebrum and cerebellum during exposure of rats to MMC. (A) RT-PCR products for nNOS, iNOS and β-actin over a 14-day time course; (B) quantification of the RT-PCR products from the experiments illustrated in A (nNOS, closed square; β-actin, open square). Extraction of total RNA and RT-PCR were performed under the conditions described in Section 2. C and P indicate control and paralysis, respectively. Each point is the average of two determinations. The alteration shown in A is a representative of data from four other animals.</note><note type="content">Fig. 3: Immunoblot analysis of protein levels of NOS isozymes in the cerebrum and cerebellum during exposure of rats to MMC. (A) protein that react with specific antibodies against nNOS and iNOS over a 14-day time course; (B) quantification of the immunoreactive proteins from the experiments illustrated in A. Electrophoresis and Western blot analysis were performed under conditions described in Section 2. C and P indicate control and paralysis, respectively. Each point is the average of two determinations. Purified mouse macrophage iNOS was used as the standard. The alteration shown in A is a representative of data from four other animals.</note><note type="content">Fig. 4: Effects of mercury derivatives on NOS activity in rat cerebellum 20,000×g supernatant. Rat cerebellum 20,000×g supernatants were incubated with a different concentration of MC (closed square), MMC (closed circle), EMC (open circle), PMC (open square) or DMM (closed triangle). Reaction mixtures (0.1 ml) containing enzyme preparation (70–116 μg of protein), 40 mM HEPES (pH 7.6) in the absence and presence of mercury compounds were preincubated at 37°C for 5 min. Mercury compounds were dissolved with different solvents as follows: MC, MMC and EMC, water; PMC, dimethyl sulfoxide (final conc., 2%); DMM, ethanol (final conc., 2%). Data indicate relative inhibition to control NOS activities with water, dimethyl sulfoxide and ethanol as solvent. Each data is the mean of duplicate experiments.</note><note type="content">Fig. 5: Elution patterns of rat cerebellum nNOS from Affi-Gel 501 column. The 20,000×g supernatant of treated rat cerebellum, following Econo-Pac 10 DG column chromatography (2.0 mg of protein) was applied to the Affi-Gel 501 column (2.0×1.0 cm, i.d.) and bound proteins were successively eluted by 5 vol. of 0.5 M NaCl (a), followed by 0.05% Tween 20 (b), 10 mM histidine (c), and 10 mM 2-ME (d) in the 50 mM Tris–HCl (pH 7.4)–0.1 mM EDTA–0.1 mM EGTA–1 μM pepstatin A–2 μM leupeptin–1 mM PMSF. Each fraction (2.1 ml) collected was employed to determine enzyme activity and to conduct immunoblot analysis. Solid lines and closed squares indicate absorbance at 280 nm and NOS activity, respectively. The eluates were electrophoresed by SDS-PAGE, followed by Western blotting by the method describing Section 2. The bands in the figure indicate; Lane 1, rat nNOS (43 ng); lane 2, macrophage iNOS (42 ng); lane 3, fraction 25 (4.1 μg).</note><note type="content">Table 1: Changes in total mercury concentration in the blood, cerebrum and cerebellum from rat subcutaneously injected with MMC (10 mg kg−1 day−1) for 8 days</note><note type="content">Table 2: Effects of pre- and post-treatment with SH compounds on the inhibition of nNOS activity caused by MC and MMC</note><subject lang="en"><genre>Keywords</genre><topic>Nitric oxide synthase</topic><topic>Enzyme activity</topic><topic>Methylmercuric chloride</topic><topic>Mercuric chloride</topic><topic>Thiol compounds</topic><topic>Inhibition</topic></subject><relatedItem type="host"><titleInfo><title>Brain Research</title></titleInfo><titleInfo type="abbreviated"><title>BRES</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1998</dateIssued></originInfo><identifier type="ISSN">0006-8993</identifier><identifier type="PII">S0006-8993(00)X0167-9</identifier><part><date>1998</date><detail type="volume"><number>798</number><caption>vol.</caption></detail><detail type="issue"><number>1–2</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>342</end></extent><extent unit="pages"><start>147</start><end>155</end></extent></part></relatedItem><identifier type="istex">DF9D5B1F6BF3A2ECD438635FB4C0C901141D6D12</identifier><identifier type="ark">ark:/67375/6H6-N6Z0091G-T</identifier><identifier type="DOI">10.1016/S0006-8993(98)00400-4</identifier><identifier type="PII">S0006-8993(98)00400-4</identifier><identifier type="ArticleID">14633</identifier><accessCondition type="use and reproduction" contentType="copyright">©1998 Elsevier Science B.V.</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science B.V., ©1998</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-N6Z0091G-T/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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