Adenosine Triphosphate-Dependent Transport of Estradiol–17β(β– D –Glucuronide) in Membrane Vesicles by Mdr1 Expressed in Insect Cells

Abstract

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) (E₂17G). MDR1 substrates/modulators inhibit adenosine triphosphate (ATP)–dependent transport of E₂17G in the rat canalicular membrane and protect against E₂17G–mediated cholestasis in isolated perfused rat liver. The present studies were designed to determine if E₂ 17G is a substrate for MDR1 using a baculovirus expression system and if other estrogen glucuronides interact with MDR1. ATP–dependent transport of E₂17G (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 E₂ 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) (E₃17G) and estriol–16α(β–d–glucuronide) (E₃16G), inhibited MDR1–mediated E₂17G transport by 58% and 35%, respectively. In contrast, noncholestatic glucuronides, estradiol–3–(β–d–glucuronide) (E₂3G) or estradiol–3–sulfate–17β(β–d–glucuronide) (E₂3SO₄17G), had no effect. E₂17G 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 E₃16G and E₃17G in isolated perfused rat liver. These studies identify E₂17G, and probably E₃16G and E₃17G, as endogenous substrates for MDR1.