Interaction of Progestins With the Human Multidrug Resistance-Associated Protein 2 (Mrp2)

Abstract

ABSTRACT:Progestins are widely used as oral contraceptives and hormone replacement therapy. Recently it has been demonstrated that many progestins are inhibitors of P-glycoprotein, possibly explaining gender differences in drug actions. In vitro evidence suggested that at least norgestimate might also inhibit other transporters like the multidrug resistance-associated protein 2 (MRP2). We therefore investigated whether norgestimate, desogestrel, medroxyprogesterone acetate, norethisterone, progesterone, cyproterone acetate, chlormadinone acetate, and levonorgestrel inhibit MRP2 in vitro using confocal laser scanning microscopy and 5-chloromethylfluorescein diacetate as a prodrug of the fluorescent 5-chloromethylfluorescein (CMF), which is actively transported by MRP2 as glutathione conjugate. Of the progestins tested, only norgestimate (50 M) and progesterone (100 M) significantly increased intracellular CMF fluorescence by 62% and 53%, respectively. In conclusion, the progestins norgestimate and progesterone significantly inhibit the transport activity of MRP2 in vitro.Multidrug resistance-associated proteins (MRPs) are a subfamily of the ATP-binding cassette transport protein family involved in drug resistance (Schinkel and Jonker, 2003). MRP2 is a key member of this group and was identified in the canalicular membrane of hepatocytes as a transporter for organic anions extruding a wide range of glutathione, glucuronate, and sulfate conjugates into the bile (Ishikawa 1993;Ito et al., 1997;Paulusma and Oude Elferink, 1997). It was also detected in renal brush-border membranes, the intestine (Schaub et al., 1997), placenta (St.-Pierre et al., 2000;Gerk and Vore, 2002), and brain (Török et al., 2003). In human carcinoma, MRP2 may confer resistance to chemotherapy (Norris et al., 1996;Nies et al., 2001;Young et al., 2001;Schinkel and Jonker, 2003), and it appears also to play a role in drug-drug interactions (Fromm et al., 2000;Bramow et al., 2001;Bode et al., 2002;Huisman et al., 2002;Giessmann et al., 2004).In recent years it has been suggested that gender could alter the activity of drug transporters like P-glycoprotein (P-gp) (Cummins et al., 2002). Hence in a previous study we have investigated whether P-gp is inhibited by progestins used in oral contraceptives and hormone replacement therapy. All progestins tested had P-gp inhibitor properties, with some of them being as potent as the well known P-gp inhibitor quinidine (Fröhlich et al., 2004). Interestingly, one of the tested progestins (norgestimate) revealed an inhibitory effect on the transport of calcein-acetoxymethylester (calcein-AM) not only in the P-gp-overexpressing cell line L-MDR1, but also in the parental cell line LLC-PK1, which only expresses low amounts of P-gp (Decorti et al., 2001;Weiss et al., 2003;Fröhlich et al., 2004), indicating that this effect was not selective. Because LLC-PK1 expresses MRP2 (Evers et al., 1996;Decorti et al., 2001) and MRP2 can transport calcein-AM (Evers et al., 2000), these data may suggest that norgestimate also...

Inhibition of MRP1/ABCC1, MRP2/ABCC2, and MRP3/ABCC3 by Nucleoside, Nucleotide, and Non-Nucleoside Reverse Transcriptase Inhibitors

“…CMFDA in a final concentration of 50 nM was subsequently added and incubated for 10 min. Data were analyzed according to Lindenmaier et al (2005). The experiments were performed at least in triplicate on different days.…”

“…Intracellular accumulation of the MF-SG in cells was analyzed with a DM IRE 2 TCS SP II confocal laser-scanning microscope (cLSM) from Leica (Bensheim, Germany) as published previously (Lindenmaier et al, 2005) with minor alterations. In brief, living cells (6 ϫ 10 5 ) were seeded on coverslips in a closed miniperfusion chamber (H. Saur, Reutlingen, Germany) directly before the experiment and preincubated for 30 min with or without the test compound in darkness at 37°C in 1 ml of transport buffer consisting of Hank's balanced salt solution and 1 mM pyruvate (Invitrogen) for energy supply.…”

“…The method for measuring the functional activity of MRP was adopted from the CMFDA assay for MRP2 (Bogman et al, 2003;Lindenmaier et al, 2005) and extended to MRP1 and MRP3. The nonfluorescent lipophilic CMFDA (MobiTec, Göttingen, Germany) passively penetrates the plasma membrane.…”

“…Inside the cells, cytosolic esterases cleave off its acetate residues, thereby releasing the fluorescent and membrane-impermeable product 5-chloromethylfluorescein (CMF), which can react (e.g., with glutathione) to form fluorescent conjugates. This methylfluorescein-glutathione complex (MF-SG) is then actively secreted by MRP2 (Bogman et al, 2003) and also by MRP1 and MRP3 if present.Intracellular accumulation of the MF-SG in cells was analyzed with a DM IRE 2 TCS SP II confocal laser-scanning microscope (cLSM) from Leica (Bensheim, Germany) as published previously (Lindenmaier et al, 2005) with minor alterations. In brief, living cells (6 ϫ 10 5 ) were seeded on coverslips in a closed miniperfusion chamber (H. Saur, Reutlingen, Germany) directly before the experiment and preincubated for 30 min with or without the test compound in darkness at 37°C in 1 ml of transport buffer consisting of Hank's balanced salt solution and 1 mM pyruvate (Invitrogen) for energy supply.…”

Antioxidant Activity of Traditional Siddha Formulation on Ccl4 Induced Liver Fibrosis in Rats

“…Glutathione reductase activity was measured by the method of Maron et al, [29] Vitamin E was estimated by the method of Quaife and Dju [30] Vitamin C was estimated by the method of Omage et al [31]. Reduced glutathione was measured by the method of Linden Maier et al [32].…”