title: Determination of p-glycoprotein ATPase activity using luciferase.
abstract: We investigated whether P-glycoprotein (P-gp) ATPase activity of Caco-2 cell membranes could be estimated by measuring consumption of ATP using luciferin-luciferase reaction, and whether the results would be useful for assessment of the interactions between P-gp and drugs. The vanadate-sensitive ATPase activity of Caco-2 cell membranes was measured rapidly with high sensitivity using luciferin-luciferase reaction. Cyclosporin A, verapamil, digoxin and quinidine stimulated the ATPase activity concentration-dependently with Km values of 5.3, 0.9, 1.2 and 4.1 microM, respectively. These values except for digoxin were comparable with previous reports. The ATPase activity and P-gp mRNA expression in Caco-2 cells were induced by all-trans-retinoic acid, digoxin and levothyroxine, but not dexamethasone or rifampicin. This method was useful to assess interactions with P-gp and drugs, and was used to elucidate the mechanisms of interaction of levothyroxine and digoxin.
authors.affiliation: Division of Pharmacy, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Tamihide
authors.initials: T
authors.lastname: Matsunaga
authors.forename: Eiji
authors.initials: E
authors.lastname: Kose
authors.forename: Sachiyo
authors.initials: S
authors.lastname: Yasuda
authors.forename: Hirohiko
authors.initials: H
authors.lastname: Ise
authors.forename: Uichi
authors.initials: U
authors.lastname: Ikeda
authors.forename: Shigeru
authors.initials: S
authors.lastname: Ohmori
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004077:Digoxin / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008156:Luciferases; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D012293:Rifampin / Q000494:pharmacology; D013974:Thyroxine / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005938:Glucocorticoids; D012333:RNA, Messenger; D004077:Digoxin; D003907:Dexamethasone; D000255:Adenosine Triphosphate; D008156:Luciferases; D000251:Adenosine Triphosphatases; D013974:Thyroxine; D012293:Rifampin