title: Mouse Bsep ATPase assay: a nonradioactive tool for assessment of the cholestatic potential of drugs.
abstract: 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.
authors.affiliation: Solvo Biotechnology, Szeged, Hungary.
authors.forename: Emese
authors.initials: E
authors.lastname: Kis
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Rajnai
authors.forename: Eniko
authors.initials: E
authors.lastname: Ioja
authors.forename: Krisztina
authors.initials: K
authors.lastname: Herédi Szabó
authors.forename: Tünde
authors.initials: T
authors.lastname: Nagy
authors.forename: Dóra
authors.initials: D
authors.lastname: Méhn
authors.forename: Péter
authors.initials: P
authors.lastname: Krajcsi
mesh_terms: 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
chemical_list: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490549:Abcb11 protein, mouse; D002784:Cholesterol; D000251:Adenosine Triphosphatases