Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells.
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.
Takahashi
Kyohei
K
Hokkaido Pharmaceutical University School of Pharmacy, 7-1 Katsuraoka-cho, Otaru, Hokkaido 047-0264, Japan.
Shibata
Tomohito
T
Oba
Tatsuya
T
Ishikawa
Tetsuya
T
Yoshikawa
Masahito
M
Tatsunami
Ryosuke
R
Takahashi
Kazuhiko
K
Tampo
Yoshiko
Y
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*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011422:Propionates; D011804:Quinolines; D013481:Superoxides; C059141:verlukast; D024483:Vitamin K 3; D005978:Glutathione