Abstract
The multidrug resistance protein 2 (MRP2/ABCC2) mediates the biliary excretion of glucuronide and glutathione conjugates of endogenous and exogenous compounds. We examined the activation of human MRP2-mediated ATP-dependent transport by the choleretic bile salt ursodeoxycholic acid (UDC) and its taurine and glycine amidates in Sf9 cell membranes expressing MRP2 using β-estradiol 17-(β-d-glucuronide) (E217G) and β-estradiol 3-(β-d-glucuronide) (E23G) as substrates. MRP2 transported E23G via classic Michaelis-Menten kinetics (Km = 122 μM; Vmax = 3.0 nmol/mg/min), whereas E217G transport showed positive cooperativity (Hill slope, 2.15; Km = 75 μM; Vmax = 3.8 nmol/mg/min). UDC, tauroursodeoxycholate, and glycoursodeoxycholate (80–100 μM) maximally stimulated E23G transport 9-, 7.9-, and 3.6-fold, respectively, whereas higher concentrations (1–2 mM) inhibited transport. At low (0.3 μM) concentrations, tauroursodeoxycholate was transported only in the presence of E217G or E23G, but not other MRP2 substrates such as methotrexate, leukotriene C4, or S-methylglutathione. Kinetic analysis of higher concentrations of tauroursodeoxycholate transport by MRP2 showed positive cooperativity (Hill slope, 1.84; Km = 127 μM; Vmax = 779 pmol/mg/min). Taurocholate (2–100 μM) was not detectably transported by MRP2 either alone or in the presence of E217G but was transported in the presence of E23G. Thus, UDC, tauroursodeoxycholate, and glycoursodeoxycholate activated MRP2 transport. Tauroursodeoxycholate was transported by MRP2 and demonstrated positive cooperativity, identifying it as the second MRP2 substrate able to stimulate its own transport. The data suggest MRP2 binding sites that can require specific complementarities between substrates and modulators of MRP2-mediated transport.
Footnotes
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This work was supported by United States Public Health Service Grants GM 55343 (to M.V.) and T32 HD07436 (to P.M.G.) and by an unrestricted gift from Axcan Pharma, Inc.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.106922.
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ABBREVIATIONS: MRP, multidrug resistance protein; ABC, ATP-binding cassette; E217G, β-estradiol 17-(β-d-glucuronide); UDC, ursodiol (ursodeoxycholic acid); GUDC, glycoursodeoxycholate; TUDC, tauroursodeoxycholate; BSEP, bile salt export pump; PKC, protein kinase C; E23G, β-estradiol 3-(β-d-glucuronide); TC, taurocholate; EV, empty vector.
- Received August 5, 2006.
- Accepted November 14, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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