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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
5
|
| pubmed:dateCreated |
1998-9-24
|
| pubmed:abstractText |
1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.
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| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Adenylate Cyclase,
http://linkedlifedata.com/resource/pubmed/chemical/Cholesterol,
http://linkedlifedata.com/resource/pubmed/chemical/Cimetidine,
http://linkedlifedata.com/resource/pubmed/chemical/Cyclic AMP,
http://linkedlifedata.com/resource/pubmed/chemical/Cyclodextrins,
http://linkedlifedata.com/resource/pubmed/chemical/Epitopes,
http://linkedlifedata.com/resource/pubmed/chemical/Guanidines,
http://linkedlifedata.com/resource/pubmed/chemical/Histamine H2 Antagonists,
http://linkedlifedata.com/resource/pubmed/chemical/Histidine,
http://linkedlifedata.com/resource/pubmed/chemical/Oligonucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Piperidines,
http://linkedlifedata.com/resource/pubmed/chemical/Ranitidine,
http://linkedlifedata.com/resource/pubmed/chemical/Receptors, Histamine H2,
http://linkedlifedata.com/resource/pubmed/chemical/aminopotentidine,
http://linkedlifedata.com/resource/pubmed/chemical/beta-Cyclodextrins,
http://linkedlifedata.com/resource/pubmed/chemical/betadex,
http://linkedlifedata.com/resource/pubmed/chemical/tiotidine
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Nov
|
| pubmed:issn |
0007-1188
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:volume |
122
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| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
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| pubmed:pagination |
867-74
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| pubmed:dateRevised |
2008-11-20
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| pubmed:meshHeading |
pubmed-meshheading:9384502-Adenylate Cyclase,
pubmed-meshheading:9384502-Animals,
pubmed-meshheading:9384502-Baculoviridae,
pubmed-meshheading:9384502-Blotting, Western,
pubmed-meshheading:9384502-Cell Line, Transformed,
pubmed-meshheading:9384502-Cholesterol,
pubmed-meshheading:9384502-Cimetidine,
pubmed-meshheading:9384502-Cyclic AMP,
pubmed-meshheading:9384502-Cyclodextrins,
pubmed-meshheading:9384502-Epitopes,
pubmed-meshheading:9384502-Fluorescent Antibody Technique,
pubmed-meshheading:9384502-Guanidines,
pubmed-meshheading:9384502-Histamine H2 Antagonists,
pubmed-meshheading:9384502-Histidine,
pubmed-meshheading:9384502-Insects,
pubmed-meshheading:9384502-Microscopy, Confocal,
pubmed-meshheading:9384502-Oligonucleotides,
pubmed-meshheading:9384502-Piperidines,
pubmed-meshheading:9384502-Ranitidine,
pubmed-meshheading:9384502-Rats,
pubmed-meshheading:9384502-Receptors, Histamine H2,
pubmed-meshheading:9384502-Transfection,
pubmed-meshheading:9384502-beta-Cyclodextrins
|
| pubmed:year |
1997
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| pubmed:articleTitle |
Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells.
|
| pubmed:affiliation |
Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Amsterdam, The Netherlands.
|
| pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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