Mouse Bsep ATPase assay: a nonradioactive tool for assessment of the cholestatic potential of drugs

• Published in: Journal of biomolecular screening Vol. 14; no. 1; p. 10
• Main Authors: Kis, Emese, Rajnai, Zsuzsanna, Ioja, Eniko, Herédi Szabó, Krisztina, Nagy, Tünde, Méhn, Dóra, Krajcsi, Péter
• Format: Journal Article
• Language: English
• Published: United States 01.01.2009
• Subjects: Adenosine Triphosphatases - analysis; Adenosine Triphosphatases - antagonists & inhibitors; Adenosine Triphosphatases - metabolism; Animals; ATP Binding Cassette Subfamily B Member 11; ATP-Binding Cassette Transporters - analysis; ATP-Binding Cassette Transporters - antagonists & inhibitors; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Biological Transport - drug effects; Cell Line; Cholestasis - drug therapy; Cholesterol - metabolism; Cholesterol - pharmacology; Mice; Radioligand Assay; Spodoptera
• ISSN: 1087-0571; 1552-454X
• DOI: 10.1177/1087057108326145

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.