Cytostar-T scintillating microplate assay for measurement of sodium-dependent bile acid uptake in transfected HEK-293 cells.
Real-time measurements of bile acid uptake into HEK-293 cell monolayers expressing the human sodium/bile acid cotransporters have been demonstrated using Cytostar-T microplates with an integral scintillating base. In these 96-well microplates, which permits culturing and observation of adherent cell monolayers, uptake of (14)C-labeled glycocholate and taurocholate into transfected HEK-293 cells was time-dependent, sodium-stimulated, and saturable. The sodium-activated uptake of 30 microM [(14)C]glycocholate (GC) via the ileal (IBAT) and liver (LBAT) transporters was 30-40 times higher than GC uptake in a sodium-free background. In addition, ouabain inhibition of the plasma membrane Na(+), K(+)-ATPase, causing the sodium gradient to collapse, resulted in total loss of glycocholate transport. Induction of gene expression by sodium butyrate showed that the amount of labeled bile acid accumulated in the cell monolayers at steady state was a function of the total amount of transporter expressed. Uptake of labeled bile acids was inhibited both by the specific IBAT inhibitor, 2164U90, and by various bile acids. No major difference was observed between IBAT and LBAT in their specificity for the bile acids tested while the dihydroxy bile acids had the highest affinity for both the transporters studied. The Cytostar-T proximity assay has been demonstrated to be an accurate and reproducible method for monitoring specific bile acid transport in transfected mammalian cells and the results are similar to those obtained by traditional methods. We conclude that the technique is an attractive approach to the cellular study of membrane transport of radiolabeled solutes in general and suggest a role in screening and characterization of novel transport inhibitors.
Bonge
H
H
Cell Biology and Biochemistry, AstraZeneca R&D Mölndal, Mölndal, S-431 83, Sweden.
Hallén
S
S
Fryklund
J
J
Sjöström
J E
JE
D001647:Bile Acids and Salts / Q000493:pharmacokinetics*; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006000:Glycocholic Acid / Q000493:pharmacokinetics; D006801:Humans; D000960:Hypolipidemic Agents / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent; D010042:Ouabain / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D012588:Scintillation Counting; D012964:Sodium / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D027981:Symporters; D013656:Taurocholic Acid / Q000493:pharmacokinetics; D013841:Thiazepines / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
C095410:2164U90; D001647:Bile Acids and Salts; D002087:Butyrates; D002352:Carrier Proteins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D000960:Hypolipidemic Agents; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; D013841:Thiazepines; C071908:sodium-bile acid cotransporter; D012965:Sodium Chloride; D010042:Ouabain; D013656:Taurocholic Acid; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D006000:Glycocholic Acid