Adenosine triphosphate–dependent transport of estradiol‐17β(β‐ d ‐glucuronide) in membrane vesicles by MDR1 expressed in insect cells

MDR1, an ABC transporter that confers multidrug resistance in tumor cells, is constitutively expressed in normal liver canalicular membrane. Human MDR1‐expressing multidrug‐resistant cells display increased resistance to estradiol‐17β(β‐ d ‐glucuronide) (E 2 17G). MDR1 substrates/modulators inhibit adenosine triphosphate (ATP)‐dependent transport of E 2 17G in the rat canalicular membrane and protect against E 2 17G‐mediated cholestasis in isolated perfused rat liver. The present studies were designed to determine if E 2 17G is a substrate for MDR1 using a baculovirus expression system and if other estrogen glucuronides interact with MDR1. ATP‐dependent transport of E 2 17G (10 μmol/L) was linear for up to 2 minutes and yielded a rate of 45.6 pmol/min/mg protein in membrane vesicles from Sf9 cells infected with MDR1‐baculovirus. This transport was saturable ( K m = 62 μmol/L) and occurred into an osmotically sensitive space. ATP‐dependent transport of E 2 17G (10 μmol/L) was inhibited 63% by 10 μmol/L daunomycin, but not by 100 μmol/L S ‐(2,4‐dinitrophenyl)glutathione (GS‐DNP) (a substrate for canalicular multispecific organic anion transporter [cMOAT]). Glucuronide conjugates of the estrogen D‐ring (100 μmol/L), estriol‐17β(β‐ d ‐glucuronide) (E 3 17G) and estriol‐16α(β‐ d ‐glucuronide) (E 3 16G), inhibited MDR1‐mediated E 2 17G transport by 58% and 35%, respectively. In contrast, noncholestatic glucuronides, estradiol‐3‐(β‐ d ‐glucuronide) (E 2 3G) or estradiol‐3‐sulfate‐17β(β‐ d ‐glucuronide) (E 2 3SO 4 17G), had no effect. E 2 17G neither stimulated MDR1 ATPase activity nor inhibited verapamil‐stimulated ATPase activity. Infusion of 1.5 μmol/L doxorubicin or 1 μmol/L taxol protected against cholestasis induced by E 3 16G and E 3 17G in isolated perfused rat liver. These studies identify E 2 17G, and probably E 3 16G and E 3 17G, as endogenous substrates for MDR1.