title: Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells.
abstract: OBJECTIVE
  Menadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).
  
  
  METHODS
  BAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.
  
  
  RESULTS
  Intracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.
  
  
  CONCLUSIONS
  We conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.
authors.affiliation: Hokkaido Pharmaceutical University School of Pharmacy, 7-1 Katsuraoka-cho, Otaru, Hokkaido 047-0264, Japan.
authors.forename: Kyohei
authors.initials: K
authors.lastname: Takahashi
authors.forename: Tomohito
authors.initials: T
authors.lastname: Shibata
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Oba
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Masahito
authors.initials: M
authors.lastname: Yoshikawa
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Tatsunami
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Takahashi
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Tampo
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D024483:Vitamin K 3 / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011422:Propionates; D011804:Quinolines; D013481:Superoxides; C059141:verlukast; D024483:Vitamin K 3; D005978:Glutathione