Activation of Gq Proteins Coupled with 5-HT2 Receptors in Rat Cerebral Cortical Membranes Assessed by Antibody-Capture Scintillation Proximity Assay/[35S]GTPγS Binding
Background/Aims: Functional activation of Gq coupled with 5-HT 2 receptors was investigated in rat cerebral cortical membranes. Methods: Antibody-capture scintillation proximity assay (SPA)/[ 35 S]GTPγS binding with anti-Gα q antibody was performed. Results: The specific [ 35 S]GTPγS binding to Gα q...
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| Veröffentlicht in: | Pharmacology 2013-01, Vol.92 (1-2), p.2-10 |
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| creator | Odagaki, Yuji Toyoshima, Ryoichi |
| description | Background/Aims: Functional activation of Gq coupled with 5-HT 2 receptors was investigated in rat cerebral cortical membranes. Methods: Antibody-capture scintillation proximity assay (SPA)/[ 35 S]GTPγS binding with anti-Gα q antibody was performed. Results: The specific [ 35 S]GTPγS binding to Gα q was increased by 5-hydroxytryptamine (5-HT) in a concentration-dependent but unsaturable manner. The increase elicited by micromolar concentrations of 5-HT was inhibited completely by ketanserin, whereas it inhibited the response by submillimolar to millimolar concentrations of 5-HT only partially. Analysis of the concentration-dependent increases by 5-HT in the absence and presence of ketanserin, methiothepin, WAY100635, and pirenzepine clearly indicates that there are two distinct components of 5-HT-stimulated [ 35 S]GTPγS binding, one of which is a pharmacologically relevant increase elicited by lower concentrations (-30 μmol/l) of 5-HT mediated through 5-HT 2 receptors and the other is pharmacologically undefined stimulation by higher concentrations of 5-HT. When 5-HT and carbachol were added simultaneously, there was apparently lack of additivity. Conclusion: It is concluded that by means of antibody-capture SPA/[ 35 S]GTPγS binding it is possible to detect two distinct components of 5-HT-elicited activation of Gq shared by M 1 muscarinic receptors, one of which is mediated through 5-HT 2 receptors and the other is derived from unknown origin in rat cerebral cortical membranes. |
| doi_str_mv | 10.1159/000351745 |
| format | Article |
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Methods: Antibody-capture scintillation proximity assay (SPA)/[ 35 S]GTPγS binding with anti-Gα q antibody was performed. Results: The specific [ 35 S]GTPγS binding to Gα q was increased by 5-hydroxytryptamine (5-HT) in a concentration-dependent but unsaturable manner. The increase elicited by micromolar concentrations of 5-HT was inhibited completely by ketanserin, whereas it inhibited the response by submillimolar to millimolar concentrations of 5-HT only partially. Analysis of the concentration-dependent increases by 5-HT in the absence and presence of ketanserin, methiothepin, WAY100635, and pirenzepine clearly indicates that there are two distinct components of 5-HT-stimulated [ 35 S]GTPγS binding, one of which is a pharmacologically relevant increase elicited by lower concentrations (-30 μmol/l) of 5-HT mediated through 5-HT 2 receptors and the other is pharmacologically undefined stimulation by higher concentrations of 5-HT. When 5-HT and carbachol were added simultaneously, there was apparently lack of additivity. Conclusion: It is concluded that by means of antibody-capture SPA/[ 35 S]GTPγS binding it is possible to detect two distinct components of 5-HT-elicited activation of Gq shared by M 1 muscarinic receptors, one of which is mediated through 5-HT 2 receptors and the other is derived from unknown origin in rat cerebral cortical membranes.</description><identifier>ISSN: 0031-7012</identifier><identifier>EISSN: 1423-0313</identifier><identifier>DOI: 10.1159/000351745</identifier><identifier>PMID: 23860332</identifier><language>eng</language><publisher>Basel, Switzerland</publisher><subject>Animals ; Antibodies - immunology ; Biological Assay ; Carbachol - pharmacology ; Cerebral Cortex - metabolism ; GTP-Binding Protein alpha Subunits, Gq-G11 - immunology ; GTP-Binding Protein alpha Subunits, Gq-G11 - metabolism ; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism ; Male ; Original Paper ; Rats ; Rats, Sprague-Dawley ; Receptors, Serotonin, 5-HT2 - metabolism ; Serotonin - pharmacology ; Serotonin Antagonists - pharmacology ; Serotonin Receptor Agonists - pharmacology ; Sulfur Radioisotopes</subject><ispartof>Pharmacology, 2013-01, Vol.92 (1-2), p.2-10</ispartof><rights>2013 S. Karger AG, Basel</rights><rights>Copyright © 2013 S. Karger AG, Basel.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c306t-bbf1edd037b6e303808f2dfdbd6c6faa29b1ba01b322ff337c9c5042fea6a2b13</citedby><cites>FETCH-LOGICAL-c306t-bbf1edd037b6e303808f2dfdbd6c6faa29b1ba01b322ff337c9c5042fea6a2b13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,2423,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23860332$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Odagaki, Yuji</creatorcontrib><creatorcontrib>Toyoshima, Ryoichi</creatorcontrib><title>Activation of Gq Proteins Coupled with 5-HT2 Receptors in Rat Cerebral Cortical Membranes Assessed by Antibody-Capture Scintillation Proximity Assay/[35S]GTPγS Binding</title><title>Pharmacology</title><addtitle>Pharmacology</addtitle><description>Background/Aims: Functional activation of Gq coupled with 5-HT 2 receptors was investigated in rat cerebral cortical membranes. Methods: Antibody-capture scintillation proximity assay (SPA)/[ 35 S]GTPγS binding with anti-Gα q antibody was performed. Results: The specific [ 35 S]GTPγS binding to Gα q was increased by 5-hydroxytryptamine (5-HT) in a concentration-dependent but unsaturable manner. The increase elicited by micromolar concentrations of 5-HT was inhibited completely by ketanserin, whereas it inhibited the response by submillimolar to millimolar concentrations of 5-HT only partially. Analysis of the concentration-dependent increases by 5-HT in the absence and presence of ketanserin, methiothepin, WAY100635, and pirenzepine clearly indicates that there are two distinct components of 5-HT-stimulated [ 35 S]GTPγS binding, one of which is a pharmacologically relevant increase elicited by lower concentrations (-30 μmol/l) of 5-HT mediated through 5-HT 2 receptors and the other is pharmacologically undefined stimulation by higher concentrations of 5-HT. When 5-HT and carbachol were added simultaneously, there was apparently lack of additivity. Conclusion: It is concluded that by means of antibody-capture SPA/[ 35 S]GTPγS binding it is possible to detect two distinct components of 5-HT-elicited activation of Gq shared by M 1 muscarinic receptors, one of which is mediated through 5-HT 2 receptors and the other is derived from unknown origin in rat cerebral cortical membranes.</description><subject>Animals</subject><subject>Antibodies - immunology</subject><subject>Biological Assay</subject><subject>Carbachol - pharmacology</subject><subject>Cerebral Cortex - metabolism</subject><subject>GTP-Binding Protein alpha Subunits, Gq-G11 - immunology</subject><subject>GTP-Binding Protein alpha Subunits, Gq-G11 - metabolism</subject><subject>Guanosine 5'-O-(3-Thiotriphosphate) - metabolism</subject><subject>Male</subject><subject>Original Paper</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Receptors, Serotonin, 5-HT2 - metabolism</subject><subject>Serotonin - pharmacology</subject><subject>Serotonin Antagonists - pharmacology</subject><subject>Serotonin Receptor Agonists - pharmacology</subject><subject>Sulfur Radioisotopes</subject><issn>0031-7012</issn><issn>1423-0313</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo9kU1v1DAQhi0EotvCgTtCPtJDWn9snN3jNoItUhFVdzkhFPljXAxJnNoOkH_Evf-D31RXu11ppBm9embekV6E3lByRmm5PCeE8JJW8_IZmtE54wXhlD9HsyzToiKUHaHjGH9mTLBq8RIdMb4QhHM2Q_9WOrnfMjnfY2_x-g5fB5_A9RHXfhxaMPiPSz9wWVxuGb4BDUPyIWLX4xuZcA0BVJBthkNyOg-foctCDxGvYoRcBqsJr_rklDdTUcshjQHwRrsste3OOXv-dZ1L0-OSnM6_8XLzfb29_n-_wReuN66_fYVeWNlGeL3vJ-jrxw_b-rK4-rL-VK-uCs2JSIVSloIxhFdKACd8QRaWGWuUEVpYKdlSUSUJVZwxazmv9FKXZM4sSCGZovwEvd_dHYK_GyGmpnNRQ_60Bz_Ghs45E4ywSmT0dIfq4GMMYJshuE6GqaGkeQymOQST2Xf7s6PqwBzIpyQy8HYH_JLhFsIB2O8_AC_elAY</recordid><startdate>20130101</startdate><enddate>20130101</enddate><creator>Odagaki, Yuji</creator><creator>Toyoshima, Ryoichi</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20130101</creationdate><title>Activation of Gq Proteins Coupled with 5-HT2 Receptors in Rat Cerebral Cortical Membranes Assessed by Antibody-Capture Scintillation Proximity Assay/[35S]GTPγS Binding</title><author>Odagaki, Yuji ; Toyoshima, Ryoichi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c306t-bbf1edd037b6e303808f2dfdbd6c6faa29b1ba01b322ff337c9c5042fea6a2b13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Animals</topic><topic>Antibodies - immunology</topic><topic>Biological Assay</topic><topic>Carbachol - pharmacology</topic><topic>Cerebral Cortex - metabolism</topic><topic>GTP-Binding Protein alpha Subunits, Gq-G11 - immunology</topic><topic>GTP-Binding Protein alpha Subunits, Gq-G11 - metabolism</topic><topic>Guanosine 5'-O-(3-Thiotriphosphate) - metabolism</topic><topic>Male</topic><topic>Original Paper</topic><topic>Rats</topic><topic>Rats, Sprague-Dawley</topic><topic>Receptors, Serotonin, 5-HT2 - metabolism</topic><topic>Serotonin - pharmacology</topic><topic>Serotonin Antagonists - pharmacology</topic><topic>Serotonin Receptor Agonists - pharmacology</topic><topic>Sulfur Radioisotopes</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Odagaki, Yuji</creatorcontrib><creatorcontrib>Toyoshima, Ryoichi</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Pharmacology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Odagaki, Yuji</au><au>Toyoshima, Ryoichi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Activation of Gq Proteins Coupled with 5-HT2 Receptors in Rat Cerebral Cortical Membranes Assessed by Antibody-Capture Scintillation Proximity Assay/[35S]GTPγS Binding</atitle><jtitle>Pharmacology</jtitle><addtitle>Pharmacology</addtitle><date>2013-01-01</date><risdate>2013</risdate><volume>92</volume><issue>1-2</issue><spage>2</spage><epage>10</epage><pages>2-10</pages><issn>0031-7012</issn><eissn>1423-0313</eissn><abstract>Background/Aims: Functional activation of Gq coupled with 5-HT 2 receptors was investigated in rat cerebral cortical membranes. Methods: Antibody-capture scintillation proximity assay (SPA)/[ 35 S]GTPγS binding with anti-Gα q antibody was performed. Results: The specific [ 35 S]GTPγS binding to Gα q was increased by 5-hydroxytryptamine (5-HT) in a concentration-dependent but unsaturable manner. The increase elicited by micromolar concentrations of 5-HT was inhibited completely by ketanserin, whereas it inhibited the response by submillimolar to millimolar concentrations of 5-HT only partially. Analysis of the concentration-dependent increases by 5-HT in the absence and presence of ketanserin, methiothepin, WAY100635, and pirenzepine clearly indicates that there are two distinct components of 5-HT-stimulated [ 35 S]GTPγS binding, one of which is a pharmacologically relevant increase elicited by lower concentrations (-30 μmol/l) of 5-HT mediated through 5-HT 2 receptors and the other is pharmacologically undefined stimulation by higher concentrations of 5-HT. When 5-HT and carbachol were added simultaneously, there was apparently lack of additivity. Conclusion: It is concluded that by means of antibody-capture SPA/[ 35 S]GTPγS binding it is possible to detect two distinct components of 5-HT-elicited activation of Gq shared by M 1 muscarinic receptors, one of which is mediated through 5-HT 2 receptors and the other is derived from unknown origin in rat cerebral cortical membranes.</abstract><cop>Basel, Switzerland</cop><pmid>23860332</pmid><doi>10.1159/000351745</doi><tpages>9</tpages></addata></record> |
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| subjects | Animals Antibodies - immunology Biological Assay Carbachol - pharmacology Cerebral Cortex - metabolism GTP-Binding Protein alpha Subunits, Gq-G11 - immunology GTP-Binding Protein alpha Subunits, Gq-G11 - metabolism Guanosine 5'-O-(3-Thiotriphosphate) - metabolism Male Original Paper Rats Rats, Sprague-Dawley Receptors, Serotonin, 5-HT2 - metabolism Serotonin - pharmacology Serotonin Antagonists - pharmacology Serotonin Receptor Agonists - pharmacology Sulfur Radioisotopes |
| title | Activation of Gq Proteins Coupled with 5-HT2 Receptors in Rat Cerebral Cortical Membranes Assessed by Antibody-Capture Scintillation Proximity Assay/[35S]GTPγS Binding |
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