A novel technique for visualizing the intracellular localization and distribution of transported polyamines in cultured pulmonary artery smooth muscle cells
The use of a combination of monofluorescein adducts of spermidine (FL-SPD) and spermine (FL-SPM) with confocal laser scanning microscopy (CLSM) provides a useful means for monitoring the fate and time-dependent changes in the distribution of transported polyamines within living cells. Polyamine-fluo...
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| Veröffentlicht in: | Journal of pharmaceutical and biomedical analysis 1998-06, Vol.17 (2), p.307-320 |
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| creator | Aziz, Shewan M Yatin, Mustafa Worthen, David R Lipke, David W Crooks, Peter A |
| description | The use of a combination of monofluorescein adducts of spermidine (FL-SPD) and spermine (FL-SPM) with confocal laser scanning microscopy (CLSM) provides a useful means for monitoring the fate and time-dependent changes in the distribution of transported polyamines within living cells. Polyamine-fluorescein adducts were synthesized from fluorescein isothiocyanate and the appropriate polyamine. Monofluorescein polyamine adducts (ratio 1:1) were isolated using thin layer chromatography, and the structure and molecular weight of the monofluorescein polyamine adducts were confirmed using NMR and mass spectroscopy, respectively. The covalent linkage of the fluorescent adduct moiety to SPD and SPM did not influence their rate of uptake by bovine pulmonary artery smooth muscle cells (PASMC). Similar to
14C-SPD and
14C-SPM, the rate of uptake of
14C-FL-SPD and
14C-FL-SPM in PASMC was temperature-dependent. Treatment for 24 h with difluoromethylornithine (DFMO), a selective blocker of the enzyme ornithine decarboxylase and an inducer of the polyamine transport system, significantly increased the cellular uptake of
14C-FL-SPD and
14C-FL-SPM compared to that of control cells. When compared to control cells, treatment of PASMC with the pyrrolizidine alkaloid monocrotaline for 24 h also significantly increased the cellular uptake of
14C-FL-SPD and
14C-FL-SPM. On the other hand, 24 h treatment of PASMC with a polymer of SPM, a selective blocker of the polyamine transport system, or with free spermine, markedly reduced the cellular accumulation of
14C-FL-SPD and
14C-FL-SPM. After a 20-min treatment of PASMC with FL-SPD or FL-SPM, CLSM revealed that adduct fluorescence was localized in the cytoplasm of living cells. Treatment with DFMO increased the cytoplasmic accumulation of both FL-SPD and FL-SPM. In addition, the fluorescence observed in the cytoplasm of chinese hamster ovary cells (CHO) was significantly higher than that detected in the cytoplasm of their polyamine transport deficient variants (CHOMGBG). The results of this study provide the first evidence of the utility of a novel method for visualizing the uptake, distribution, and cellular localization of transported polyamines in viable cultured mammalian cells. |
| doi_str_mv | 10.1016/S0731-7085(98)00016-8 |
| format | Article |
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14C-SPD and
14C-SPM, the rate of uptake of
14C-FL-SPD and
14C-FL-SPM in PASMC was temperature-dependent. Treatment for 24 h with difluoromethylornithine (DFMO), a selective blocker of the enzyme ornithine decarboxylase and an inducer of the polyamine transport system, significantly increased the cellular uptake of
14C-FL-SPD and
14C-FL-SPM compared to that of control cells. When compared to control cells, treatment of PASMC with the pyrrolizidine alkaloid monocrotaline for 24 h also significantly increased the cellular uptake of
14C-FL-SPD and
14C-FL-SPM. On the other hand, 24 h treatment of PASMC with a polymer of SPM, a selective blocker of the polyamine transport system, or with free spermine, markedly reduced the cellular accumulation of
14C-FL-SPD and
14C-FL-SPM. After a 20-min treatment of PASMC with FL-SPD or FL-SPM, CLSM revealed that adduct fluorescence was localized in the cytoplasm of living cells. Treatment with DFMO increased the cytoplasmic accumulation of both FL-SPD and FL-SPM. In addition, the fluorescence observed in the cytoplasm of chinese hamster ovary cells (CHO) was significantly higher than that detected in the cytoplasm of their polyamine transport deficient variants (CHOMGBG). The results of this study provide the first evidence of the utility of a novel method for visualizing the uptake, distribution, and cellular localization of transported polyamines in viable cultured mammalian cells.</description><identifier>ISSN: 0731-7085</identifier><identifier>EISSN: 1873-264X</identifier><identifier>DOI: 10.1016/S0731-7085(98)00016-8</identifier><identifier>PMID: 9638584</identifier><identifier>CODEN: JPBADA</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Animals ; Biological and medical sciences ; Cattle ; Cell physiology ; Cells, Cultured - metabolism ; Difluoromethylornithine ; Fluorescein ; Fluorescein-polyamine adducts ; Fundamental and applied biological sciences. Psychology ; Membrane and intracellular transports ; Microscopy, Confocal ; Molecular and cellular biology ; Muscle, Smooth, Vascular - metabolism ; Polyamine synthesis ; Polyamine transport ; Polymer of spermine ; Pulmonary Artery - metabolism ; Smooth muscle cells ; Spermidine - analysis ; Spermidine - metabolism ; Spermine - analysis ; Spermine - metabolism</subject><ispartof>Journal of pharmaceutical and biomedical analysis, 1998-06, Vol.17 (2), p.307-320</ispartof><rights>1998 Elsevier Science B.V.</rights><rights>1998 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c455t-b6f4640c3164889bce31a534ecdfce1e1e4b11d584d093337f4e1ddec5529f443</citedby><cites>FETCH-LOGICAL-c455t-b6f4640c3164889bce31a534ecdfce1e1e4b11d584d093337f4e1ddec5529f443</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/S0731-7085(98)00016-8$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3538,27906,27907,45977</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=2324584$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9638584$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Aziz, Shewan M</creatorcontrib><creatorcontrib>Yatin, Mustafa</creatorcontrib><creatorcontrib>Worthen, David R</creatorcontrib><creatorcontrib>Lipke, David W</creatorcontrib><creatorcontrib>Crooks, Peter A</creatorcontrib><title>A novel technique for visualizing the intracellular localization and distribution of transported polyamines in cultured pulmonary artery smooth muscle cells</title><title>Journal of pharmaceutical and biomedical analysis</title><addtitle>J Pharm Biomed Anal</addtitle><description>The use of a combination of monofluorescein adducts of spermidine (FL-SPD) and spermine (FL-SPM) with confocal laser scanning microscopy (CLSM) provides a useful means for monitoring the fate and time-dependent changes in the distribution of transported polyamines within living cells. Polyamine-fluorescein adducts were synthesized from fluorescein isothiocyanate and the appropriate polyamine. Monofluorescein polyamine adducts (ratio 1:1) were isolated using thin layer chromatography, and the structure and molecular weight of the monofluorescein polyamine adducts were confirmed using NMR and mass spectroscopy, respectively. The covalent linkage of the fluorescent adduct moiety to SPD and SPM did not influence their rate of uptake by bovine pulmonary artery smooth muscle cells (PASMC). Similar to
14C-SPD and
14C-SPM, the rate of uptake of
14C-FL-SPD and
14C-FL-SPM in PASMC was temperature-dependent. Treatment for 24 h with difluoromethylornithine (DFMO), a selective blocker of the enzyme ornithine decarboxylase and an inducer of the polyamine transport system, significantly increased the cellular uptake of
14C-FL-SPD and
14C-FL-SPM compared to that of control cells. When compared to control cells, treatment of PASMC with the pyrrolizidine alkaloid monocrotaline for 24 h also significantly increased the cellular uptake of
14C-FL-SPD and
14C-FL-SPM. On the other hand, 24 h treatment of PASMC with a polymer of SPM, a selective blocker of the polyamine transport system, or with free spermine, markedly reduced the cellular accumulation of
14C-FL-SPD and
14C-FL-SPM. After a 20-min treatment of PASMC with FL-SPD or FL-SPM, CLSM revealed that adduct fluorescence was localized in the cytoplasm of living cells. Treatment with DFMO increased the cytoplasmic accumulation of both FL-SPD and FL-SPM. In addition, the fluorescence observed in the cytoplasm of chinese hamster ovary cells (CHO) was significantly higher than that detected in the cytoplasm of their polyamine transport deficient variants (CHOMGBG). The results of this study provide the first evidence of the utility of a novel method for visualizing the uptake, distribution, and cellular localization of transported polyamines in viable cultured mammalian cells.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Cattle</subject><subject>Cell physiology</subject><subject>Cells, Cultured - metabolism</subject><subject>Difluoromethylornithine</subject><subject>Fluorescein</subject><subject>Fluorescein-polyamine adducts</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Membrane and intracellular transports</subject><subject>Microscopy, Confocal</subject><subject>Molecular and cellular biology</subject><subject>Muscle, Smooth, Vascular - metabolism</subject><subject>Polyamine synthesis</subject><subject>Polyamine transport</subject><subject>Polymer of spermine</subject><subject>Pulmonary Artery - metabolism</subject><subject>Smooth muscle cells</subject><subject>Spermidine - analysis</subject><subject>Spermidine - metabolism</subject><subject>Spermine - analysis</subject><subject>Spermine - metabolism</subject><issn>0731-7085</issn><issn>1873-264X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU2LFDEQhoMo6-zqT1jIQWT30NrpJN3pkyyLX7DgQQVvIZ1UO5F0MuZjYP0t_ljTM8NcJYeCqqdSVe-L0DVp35CW9G-_tgMlzdAKfjOK27atuUY8QRsiBtp0PfvxFG3OyHN0mdKvCnEysgt0MfZUcME26O8d9mEPDmfQW29_F8BziHhvU1HO_rH-J85bwNbnqDQ4V5yK2AW9FlW2wWPlDTY25WinckiEGVfYp12IGQzeBfeoFush1V-wLi6XuKaLW4JX8RGritWQlhDyFi8laQd4nZVeoGezcglenuIV-v7h_bf7T83Dl4-f7-8eGs04z83Uz6xnraakZ0KMkwZKFKcMtJk1kPrYRIip95p2pJQOMwNiDGjOu3FmjF6h18d_dzFUBVKWi03rBspDKEkO48jF0PEK8iOoY0gpwix30S71CElauboiD67IVXI5CnlwRYrad30aUKYFzLnrZEOtvzrVVarSzlU-bdMZ62jHjti7IwZVjL2FKJO24DUYG0FnaYL9zyL_AA4NrrE</recordid><startdate>19980601</startdate><enddate>19980601</enddate><creator>Aziz, Shewan M</creator><creator>Yatin, Mustafa</creator><creator>Worthen, David R</creator><creator>Lipke, David W</creator><creator>Crooks, Peter A</creator><general>Elsevier B.V</general><general>Elsevier Science</general><scope>IQODW</scope><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>19980601</creationdate><title>A novel technique for visualizing the intracellular localization and distribution of transported polyamines in cultured pulmonary artery smooth muscle cells</title><author>Aziz, Shewan M ; Yatin, Mustafa ; Worthen, David R ; Lipke, David W ; Crooks, Peter A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c455t-b6f4640c3164889bce31a534ecdfce1e1e4b11d584d093337f4e1ddec5529f443</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Cattle</topic><topic>Cell physiology</topic><topic>Cells, Cultured - metabolism</topic><topic>Difluoromethylornithine</topic><topic>Fluorescein</topic><topic>Fluorescein-polyamine adducts</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Membrane and intracellular transports</topic><topic>Microscopy, Confocal</topic><topic>Molecular and cellular biology</topic><topic>Muscle, Smooth, Vascular - metabolism</topic><topic>Polyamine synthesis</topic><topic>Polyamine transport</topic><topic>Polymer of spermine</topic><topic>Pulmonary Artery - metabolism</topic><topic>Smooth muscle cells</topic><topic>Spermidine - analysis</topic><topic>Spermidine - metabolism</topic><topic>Spermine - analysis</topic><topic>Spermine - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Aziz, Shewan M</creatorcontrib><creatorcontrib>Yatin, Mustafa</creatorcontrib><creatorcontrib>Worthen, David R</creatorcontrib><creatorcontrib>Lipke, David W</creatorcontrib><creatorcontrib>Crooks, Peter A</creatorcontrib><collection>Pascal-Francis</collection><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 pharmaceutical and biomedical analysis</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Aziz, Shewan M</au><au>Yatin, Mustafa</au><au>Worthen, David R</au><au>Lipke, David W</au><au>Crooks, Peter A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A novel technique for visualizing the intracellular localization and distribution of transported polyamines in cultured pulmonary artery smooth muscle cells</atitle><jtitle>Journal of pharmaceutical and biomedical analysis</jtitle><addtitle>J Pharm Biomed Anal</addtitle><date>1998-06-01</date><risdate>1998</risdate><volume>17</volume><issue>2</issue><spage>307</spage><epage>320</epage><pages>307-320</pages><issn>0731-7085</issn><eissn>1873-264X</eissn><coden>JPBADA</coden><abstract>The use of a combination of monofluorescein adducts of spermidine (FL-SPD) and spermine (FL-SPM) with confocal laser scanning microscopy (CLSM) provides a useful means for monitoring the fate and time-dependent changes in the distribution of transported polyamines within living cells. Polyamine-fluorescein adducts were synthesized from fluorescein isothiocyanate and the appropriate polyamine. Monofluorescein polyamine adducts (ratio 1:1) were isolated using thin layer chromatography, and the structure and molecular weight of the monofluorescein polyamine adducts were confirmed using NMR and mass spectroscopy, respectively. The covalent linkage of the fluorescent adduct moiety to SPD and SPM did not influence their rate of uptake by bovine pulmonary artery smooth muscle cells (PASMC). Similar to
14C-SPD and
14C-SPM, the rate of uptake of
14C-FL-SPD and
14C-FL-SPM in PASMC was temperature-dependent. Treatment for 24 h with difluoromethylornithine (DFMO), a selective blocker of the enzyme ornithine decarboxylase and an inducer of the polyamine transport system, significantly increased the cellular uptake of
14C-FL-SPD and
14C-FL-SPM compared to that of control cells. When compared to control cells, treatment of PASMC with the pyrrolizidine alkaloid monocrotaline for 24 h also significantly increased the cellular uptake of
14C-FL-SPD and
14C-FL-SPM. On the other hand, 24 h treatment of PASMC with a polymer of SPM, a selective blocker of the polyamine transport system, or with free spermine, markedly reduced the cellular accumulation of
14C-FL-SPD and
14C-FL-SPM. After a 20-min treatment of PASMC with FL-SPD or FL-SPM, CLSM revealed that adduct fluorescence was localized in the cytoplasm of living cells. Treatment with DFMO increased the cytoplasmic accumulation of both FL-SPD and FL-SPM. In addition, the fluorescence observed in the cytoplasm of chinese hamster ovary cells (CHO) was significantly higher than that detected in the cytoplasm of their polyamine transport deficient variants (CHOMGBG). The results of this study provide the first evidence of the utility of a novel method for visualizing the uptake, distribution, and cellular localization of transported polyamines in viable cultured mammalian cells.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>9638584</pmid><doi>10.1016/S0731-7085(98)00016-8</doi><tpages>14</tpages></addata></record> |
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| ispartof | Journal of pharmaceutical and biomedical analysis, 1998-06, Vol.17 (2), p.307-320 |
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| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Animals Biological and medical sciences Cattle Cell physiology Cells, Cultured - metabolism Difluoromethylornithine Fluorescein Fluorescein-polyamine adducts Fundamental and applied biological sciences. Psychology Membrane and intracellular transports Microscopy, Confocal Molecular and cellular biology Muscle, Smooth, Vascular - metabolism Polyamine synthesis Polyamine transport Polymer of spermine Pulmonary Artery - metabolism Smooth muscle cells Spermidine - analysis Spermidine - metabolism Spermine - analysis Spermine - metabolism |
| title | A novel technique for visualizing the intracellular localization and distribution of transported polyamines in cultured pulmonary artery smooth muscle cells |
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