Mouse Bsep ATPase assay: a nonradioactive tool for assessment of the cholestatic potential of drugs.
The mouse ortholog of the human bile salt export pump (BSEP) transporter was expressed in a baculovirus-infected insect cell (Sf9) system to study the effect of membrane cholesterol content on the transporter function. The transport activity of cholesterol-loaded mouse Bsep-HAM-Sf9 vesicles was determined in a vesicular transport assay with taurochenodeoxycholate (TCDC), a known BSEP substrate. Mouse Bsep transports TCDC at a high rate that can be sensitively detected in the ATPase assay. Cholesterol upload of the Sf9 membrane potentiates both TCDC transport and TCDC-stimulated ATPase activities. Inhibitory effect of BSEP interactors on probe substrate transport was tested in both vesicular transport and ATPase assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC(50) values measured in TCDC-stimulated mBsep ATPase assay and in the human BSEP vesicular transport assay utilizing taurocholate (TC) as probe substrate. This upgraded form of the mouse Bsep-HAM ATPase assay is a user friendly, sensitive, nonradioactive method for early high-throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular transport inhibition assay.
Kis
Emese
E
Solvo Biotechnology, Szeged, Hungary.
Rajnai
Zsuzsanna
Z
Ioja
Eniko
E
Herédi Szabó
Krisztina
K
Nagy
Tünde
T
Méhn
Dóra
D
Krajcsi
Péter
P
D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002779:Cholestasis / Q000188:drug therapy; D002784:Cholesterol / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D011869:Radioligand Assay; D018411:Spodoptera
D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490549:Abcb11 protein, mouse; D002784:Cholesterol; D000251:Adenosine Triphosphatases