Characterization of G-protein Signaling in Ventricular Myocytes From the Adult Mouse Heart: Differences From the Rat
We have developed a high yield technique for isolating ventricular myocytes from adult mouse hearts. This collagenase-trypsin procedure yields 3–6×106cells/heart. The cells are rod-shaped, roughly 20μM ×100 μM and Ca++tolerant, with viability of 65–80%. Binding studies with [125I]ICYP demonstrate th...
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Veröffentlicht in: | Journal of molecular and cellular cardiology 2000-07, Vol.32 (7), p.1211-1221 |
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description | We have developed a high yield technique for isolating ventricular myocytes from adult mouse hearts. This collagenase-trypsin procedure yields 3–6×106cells/heart. The cells are rod-shaped, roughly 20μM ×100 μM and Ca++tolerant, with viability of 65–80%. Binding studies with [125I]ICYP demonstrate the presence of β -adrenergic receptors at a density of 83 fmol/mg membrane protein. Assessment of the effects of the β1-specific antagonist CGP 20712A on [125I]ICYP binding and on isoproterenol (ISO)-sensitive adenylyl cyclase activity indicates that 67% of the receptors are β1and 33% are β2, compared to 16–20%β2in rat myocytes. Mouse myocytes respond to isoproterenol to produce cyclic AMP with an EC50≈110±20 n M. A functional Gipathway is demonstrated by inhibition of ISO-stimulated cyclic AMP accumulation by endothelin, carbachol and ATP and by sensitivity of this inhibition to pertussis toxin. As assessed by inositol phosphate production, endothelin and ATP stimulate the activity of the Gq-phospholipase C pathway, whereas carbachol, PGF2 αand α1-adrenergic receptor agonists show no significant effect. The inability of α1-adrenergic receptor agonists to induce phosphoinositide hydrolysis in mouse myocytes differs from a several fold α1-adrenergic activation that occurs in rat. Biochemical and pharmacological profiles, as well as the need for modifications in experimental design, indicate that mouse myocytes differ substantially from rat cardiac myocytes. |
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This collagenase-trypsin procedure yields 3–6×106cells/heart. The cells are rod-shaped, roughly 20μM ×100 μM and Ca++tolerant, with viability of 65–80%. Binding studies with [125I]ICYP demonstrate the presence of β -adrenergic receptors at a density of 83 fmol/mg membrane protein. Assessment of the effects of the β1-specific antagonist CGP 20712A on [125I]ICYP binding and on isoproterenol (ISO)-sensitive adenylyl cyclase activity indicates that 67% of the receptors are β1and 33% are β2, compared to 16–20%β2in rat myocytes. Mouse myocytes respond to isoproterenol to produce cyclic AMP with an EC50≈110±20 n M. A functional Gipathway is demonstrated by inhibition of ISO-stimulated cyclic AMP accumulation by endothelin, carbachol and ATP and by sensitivity of this inhibition to pertussis toxin. As assessed by inositol phosphate production, endothelin and ATP stimulate the activity of the Gq-phospholipase C pathway, whereas carbachol, PGF2 αand α1-adrenergic receptor agonists show no significant effect. The inability of α1-adrenergic receptor agonists to induce phosphoinositide hydrolysis in mouse myocytes differs from a several fold α1-adrenergic activation that occurs in rat. Biochemical and pharmacological profiles, as well as the need for modifications in experimental design, indicate that mouse myocytes differ substantially from rat cardiac myocytes.</description><identifier>ISSN: 0022-2828</identifier><identifier>EISSN: 1095-8584</identifier><identifier>DOI: 10.1006/jmcc.2000.1156</identifier><identifier>PMID: 10860764</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>Adenylate Cyclase Toxin ; Adenylyl Cyclases - metabolism ; Adrenergic beta-Agonists - pharmacology ; Animals ; Cell Culture Techniques - methods ; Cells, Cultured ; Cholinergic Agonists - pharmacology ; Cyclic AMP - metabolism ; Dose-Response Relationship, Drug ; GTP-Binding Proteins - metabolism ; Heart - physiology ; Inositol Phosphates - biosynthesis ; Isoproterenol - pharmacology ; Male ; Mice ; Myocardium - cytology ; Myocardium - metabolism ; Pertussis Toxin ; Phosphoinositide metabolism ; Protein Binding ; Rats ; Receptors, Adrenergic, beta - metabolism ; Signal Transduction ; Transgenic mice β -adrenergic receptors α1-adrenergic receptors ; Virulence Factors, Bordetella - pharmacology</subject><ispartof>Journal of molecular and cellular cardiology, 2000-07, Vol.32 (7), p.1211-1221</ispartof><rights>2000 Academic Press</rights><rights>Copyright 2000 Academic Press.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c406t-552ae07212d8b02b3bcd78d231cca458351bb7be64c75ebffbe7a744cd6960603</citedby><cites>FETCH-LOGICAL-c406t-552ae07212d8b02b3bcd78d231cca458351bb7be64c75ebffbe7a744cd6960603</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/jmcc.2000.1156$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>315,781,785,3551,27929,27930,46000</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10860764$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hilal-Dandan, Randa</creatorcontrib><creatorcontrib>Kanter, Joan R</creatorcontrib><creatorcontrib>Brunton, Laurence L</creatorcontrib><title>Characterization of G-protein Signaling in Ventricular Myocytes From the Adult Mouse Heart: Differences From the Rat</title><title>Journal of molecular and cellular cardiology</title><addtitle>J Mol Cell Cardiol</addtitle><description>We have developed a high yield technique for isolating ventricular myocytes from adult mouse hearts. This collagenase-trypsin procedure yields 3–6×106cells/heart. The cells are rod-shaped, roughly 20μM ×100 μM and Ca++tolerant, with viability of 65–80%. Binding studies with [125I]ICYP demonstrate the presence of β -adrenergic receptors at a density of 83 fmol/mg membrane protein. Assessment of the effects of the β1-specific antagonist CGP 20712A on [125I]ICYP binding and on isoproterenol (ISO)-sensitive adenylyl cyclase activity indicates that 67% of the receptors are β1and 33% are β2, compared to 16–20%β2in rat myocytes. Mouse myocytes respond to isoproterenol to produce cyclic AMP with an EC50≈110±20 n M. A functional Gipathway is demonstrated by inhibition of ISO-stimulated cyclic AMP accumulation by endothelin, carbachol and ATP and by sensitivity of this inhibition to pertussis toxin. As assessed by inositol phosphate production, endothelin and ATP stimulate the activity of the Gq-phospholipase C pathway, whereas carbachol, PGF2 αand α1-adrenergic receptor agonists show no significant effect. The inability of α1-adrenergic receptor agonists to induce phosphoinositide hydrolysis in mouse myocytes differs from a several fold α1-adrenergic activation that occurs in rat. Biochemical and pharmacological profiles, as well as the need for modifications in experimental design, indicate that mouse myocytes differ substantially from rat cardiac myocytes.</description><subject>Adenylate Cyclase Toxin</subject><subject>Adenylyl Cyclases - metabolism</subject><subject>Adrenergic beta-Agonists - pharmacology</subject><subject>Animals</subject><subject>Cell Culture Techniques - methods</subject><subject>Cells, Cultured</subject><subject>Cholinergic Agonists - pharmacology</subject><subject>Cyclic AMP - metabolism</subject><subject>Dose-Response Relationship, Drug</subject><subject>GTP-Binding Proteins - metabolism</subject><subject>Heart - physiology</subject><subject>Inositol Phosphates - biosynthesis</subject><subject>Isoproterenol - pharmacology</subject><subject>Male</subject><subject>Mice</subject><subject>Myocardium - cytology</subject><subject>Myocardium - metabolism</subject><subject>Pertussis Toxin</subject><subject>Phosphoinositide metabolism</subject><subject>Protein Binding</subject><subject>Rats</subject><subject>Receptors, Adrenergic, beta - metabolism</subject><subject>Signal Transduction</subject><subject>Transgenic mice β -adrenergic receptors α1-adrenergic receptors</subject><subject>Virulence Factors, Bordetella - pharmacology</subject><issn>0022-2828</issn><issn>1095-8584</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kEtLxDAURoMoOj62LiUrdx2TtE1SdzI-QRF8bUOS3o6RPjRJhfHXmzIuZuPq8sG5H_cehI4pmVNC-NlHZ-2cEZIiLfkWmlFSlZksZbGNZoQwljHJ5B7aD-EjUVWR57tojxLJieDFDMXFu_baRvDuR0c39Hho8E326YcIrsfPbtnr1vVLnMIb9NE7O7ba44fVYFcRAr72Q4fjO-CLemwjfhjGAPgWtI_n-NI1DXjo7Sb3pOMh2ml0G-Dobx6g1-url8Vtdv94c7e4uM9sQXjMypJpIIJRVktDmMmNrYWsWU6t1UUp85IaIwzwwooSTNMYEFoUha15xQkn-QE6Xfemd75GCFF1LlhoW91DulMJSisueJXA-Rq0fgjBQ6M-veu0XylK1ORZTZ7V5FlNntPCyV_zaDqoN_C12ATINQDpv28HXgXrJhO182Cjqgf3X_cvCwGNTg</recordid><startdate>20000701</startdate><enddate>20000701</enddate><creator>Hilal-Dandan, Randa</creator><creator>Kanter, Joan R</creator><creator>Brunton, Laurence L</creator><general>Elsevier Ltd</general><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>20000701</creationdate><title>Characterization of G-protein Signaling in Ventricular Myocytes From the Adult Mouse Heart: Differences From the Rat</title><author>Hilal-Dandan, Randa ; Kanter, Joan R ; Brunton, Laurence L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c406t-552ae07212d8b02b3bcd78d231cca458351bb7be64c75ebffbe7a744cd6960603</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Adenylate Cyclase Toxin</topic><topic>Adenylyl Cyclases - metabolism</topic><topic>Adrenergic beta-Agonists - pharmacology</topic><topic>Animals</topic><topic>Cell Culture Techniques - methods</topic><topic>Cells, Cultured</topic><topic>Cholinergic Agonists - pharmacology</topic><topic>Cyclic AMP - metabolism</topic><topic>Dose-Response Relationship, Drug</topic><topic>GTP-Binding Proteins - metabolism</topic><topic>Heart - physiology</topic><topic>Inositol Phosphates - biosynthesis</topic><topic>Isoproterenol - pharmacology</topic><topic>Male</topic><topic>Mice</topic><topic>Myocardium - cytology</topic><topic>Myocardium - metabolism</topic><topic>Pertussis Toxin</topic><topic>Phosphoinositide metabolism</topic><topic>Protein Binding</topic><topic>Rats</topic><topic>Receptors, Adrenergic, beta - metabolism</topic><topic>Signal Transduction</topic><topic>Transgenic mice β -adrenergic receptors α1-adrenergic receptors</topic><topic>Virulence Factors, Bordetella - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hilal-Dandan, Randa</creatorcontrib><creatorcontrib>Kanter, Joan R</creatorcontrib><creatorcontrib>Brunton, Laurence L</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>Journal of molecular and cellular cardiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hilal-Dandan, Randa</au><au>Kanter, Joan R</au><au>Brunton, Laurence L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of G-protein Signaling in Ventricular Myocytes From the Adult Mouse Heart: Differences From the Rat</atitle><jtitle>Journal of molecular and cellular cardiology</jtitle><addtitle>J Mol Cell Cardiol</addtitle><date>2000-07-01</date><risdate>2000</risdate><volume>32</volume><issue>7</issue><spage>1211</spage><epage>1221</epage><pages>1211-1221</pages><issn>0022-2828</issn><eissn>1095-8584</eissn><abstract>We have developed a high yield technique for isolating ventricular myocytes from adult mouse hearts. This collagenase-trypsin procedure yields 3–6×106cells/heart. The cells are rod-shaped, roughly 20μM ×100 μM and Ca++tolerant, with viability of 65–80%. Binding studies with [125I]ICYP demonstrate the presence of β -adrenergic receptors at a density of 83 fmol/mg membrane protein. Assessment of the effects of the β1-specific antagonist CGP 20712A on [125I]ICYP binding and on isoproterenol (ISO)-sensitive adenylyl cyclase activity indicates that 67% of the receptors are β1and 33% are β2, compared to 16–20%β2in rat myocytes. Mouse myocytes respond to isoproterenol to produce cyclic AMP with an EC50≈110±20 n M. A functional Gipathway is demonstrated by inhibition of ISO-stimulated cyclic AMP accumulation by endothelin, carbachol and ATP and by sensitivity of this inhibition to pertussis toxin. As assessed by inositol phosphate production, endothelin and ATP stimulate the activity of the Gq-phospholipase C pathway, whereas carbachol, PGF2 αand α1-adrenergic receptor agonists show no significant effect. The inability of α1-adrenergic receptor agonists to induce phosphoinositide hydrolysis in mouse myocytes differs from a several fold α1-adrenergic activation that occurs in rat. Biochemical and pharmacological profiles, as well as the need for modifications in experimental design, indicate that mouse myocytes differ substantially from rat cardiac myocytes.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>10860764</pmid><doi>10.1006/jmcc.2000.1156</doi><tpages>11</tpages></addata></record> |
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subjects | Adenylate Cyclase Toxin Adenylyl Cyclases - metabolism Adrenergic beta-Agonists - pharmacology Animals Cell Culture Techniques - methods Cells, Cultured Cholinergic Agonists - pharmacology Cyclic AMP - metabolism Dose-Response Relationship, Drug GTP-Binding Proteins - metabolism Heart - physiology Inositol Phosphates - biosynthesis Isoproterenol - pharmacology Male Mice Myocardium - cytology Myocardium - metabolism Pertussis Toxin Phosphoinositide metabolism Protein Binding Rats Receptors, Adrenergic, beta - metabolism Signal Transduction Transgenic mice β -adrenergic receptors α1-adrenergic receptors Virulence Factors, Bordetella - pharmacology |
title | Characterization of G-protein Signaling in Ventricular Myocytes From the Adult Mouse Heart: Differences From the Rat |
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