Functional and histochemical analysis of MDR3 P-glycoprotein in a tetracycline-controlled gene expression system.
Aim of the present study was to establish a cell system to study the physiological function of human MDR3 P-glycoprotein in cellular phosphatidylcholine (PC) secretion. MDR3 cDNA was expressed in HeLa cells using the tet-off system together with a luciferase reporter gene. MDR3 Pgp expression was turned on upon removal of doxycycline as shown by Western blot analysis. Immunohistochemistry using a specific anti human MDR3 Pgp antibody revealed a prominent staining of MDR3 Pgp covering the cytoplasm and the area of the plasma membrane. In presence of doxycycline MDR3 Pgp expression was turned off. For analysis of PC secretory activity, MDR3 Pgp expressing and non-expressing cells as well as control HeLa cells with low endogenous MDR3 were preincubated with [(3)H]choline for synthesis of cellular [(3)H]PC. Cells were then incubated for 2 h in media with 0-4 mM taurocholate (TC) and release of cellular [(3)H]PC was recorded. [(3)H]PC secretion was observed in presence of TC without impairing cell viability. There was a significant increase in [(3)H]PC excretion in MDR3 Pgp expressing cells compared to non-expressing controls (e.g. 4.5 fold at 4 mM TC), revealing a high efficiency of transport activity (turnover). From the data it is concluded that the MDR3 Pgp expressing cell system under control of a doxycycline responsive promotor is functionally active and provides a tool to further study MDR3 Pgp mediated transport.
Fitscher
B A
BA
Department of Internal Medicine, University Hospital, University of Heidelberg, Bergheimer Str. 58, D-69115 Heidelberg, Germany.
Ehehalt
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Jochims
C
C
Pohl
J
J
Herrmann
T
T
Stremmel
W
W
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002794:Choline / Q000493:pharmacokinetics; D003001:Cloning, Molecular; D003524:Cyclosporins / Q000494:pharmacology; D018076:DNA, Complementary; D004318:Doxycycline / Q000494:pharmacology*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D006367:HeLa Cells; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008156:Luciferases / Q000235:genetics; D010713:Phosphatidylcholines / Q000378:metabolism; D011817:Rabbits; D013656:Taurocholic Acid / Q000378:metabolism; D014316:Tritium; D014700:Verapamil / Q000494:pharmacology
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D000900:Anti-Bacterial Agents; D002121:Calcium Channel Blockers; D003524:Cyclosporins; D018076:DNA, Complementary; D010713:Phosphatidylcholines; D014316:Tritium; D013656:Taurocholic Acid; C095122:multidrug resistance protein 3; D014700:Verapamil; D008156:Luciferases; D004318:Doxycycline; D002794:Choline; C070272:valspodar