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Research ArticleArticle

Cysteinyl Leukotriene Receptor 1/2 Antagonists Nonselectively Modulate Organic Anion Transport by Multidrug Resistance Proteins (MRP1-4)

Mark A. Csandl, Gwenaëlle Conseil and Susan P. C. Cole
Drug Metabolism and Disposition June 2016, 44 (6) 857-866; DOI: https://doi.org/10.1124/dmd.116.069468

Abstract

Active efflux of both drugs and organic anion metabolites is mediated by the multidrug resistance proteins (MRPs). MRP1 (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), and MRP4 (ABCC4) have partially overlapping substrate specificities and all transport 17β-estradiol 17-(β-d-glucuronide) (E217βG). The cysteinyl leukotriene receptor 1 (CysLT1R) antagonist MK-571 inhibits all four MRP homologs, but little is known about the modulatory effects of newer leukotriene modifiers (LTMs). Here we examined the effects of seven CysLT1R- and CysLT2R-selective LTMs on E217βG uptake into MRP1–4-enriched inside-out membrane vesicles. Their effects on uptake of an additional physiologic solute were also measured for MRP1 [leukotriene C4 (LTC4)] and MRP4 [prostaglandin E2 (PGE2)]. The two CysLT2R-selective LTMs studied were generally more potent inhibitors than CysLT1R-selective LTMs, but neither class of antagonists showed any MRP selectivity. For E217βG uptake, LTM IC50s ranged from 1.2 to 26.9 μM and were most comparable for MRP1 and MRP4. The LTM rank order inhibitory potencies for E217βG versus LTC4 uptake by MRP1, and E217βG versus PGE2 uptake by MRP4, were also similar. Three of four CysLT1R-selective LTMs also stimulated MRP2 (but not MRP3) transport and thus exerted a concentration-dependent biphasic effect on MRP2. The fourth CysLT1R antagonist, LY171883, only stimulated MRP2 (and MRP3) transport but none of the MRPs were stimulated by either CysLT2R-selective LTM. We conclude that, in contrast to their CysLTR selectivity, CysLTR antagonists show no MRP homolog selectivity, and data should be interpreted cautiously if obtained from LTMs in systems in which more than one MRP is present.

Introduction

Nine proteins composing a subset of the C subfamily of the ATP-binding cassette (ABC) superfamily are known as ABCC or multidrug resistance proteins (MRPs) (Slot et al., 2011). The four “short” MRPs have a four-domain structure with two nucleotide binding domains, each preceded by a membrane-spanning domain (MSD1/2). The five “long” MRPs have an additional NH2-terminally located MSD0. MSD1 and MSD2 form the pore through which solutes are translocated, powered by ATP binding and hydrolysis.

Each MRP has its own distinct, albeit somewhat overlapping, substrate specificity and tissue distribution, and their roles in human (patho)physiology and pharmacology vary accordingly (Slot et al., 2011; Keppler, 2011). The long MRP1–3 and the short MRP4 are the most pharmacologically relevant MRPs and can influence pharmacokinetics, play important roles in tissue defense, and participate in signaling pathways (Tang et al., 2013; van der Schoor et al., 2015).

Elevated MRP1/ABCC1 levels in tumor cells cause multidrug resistance, consistent with MRP1’s ability to efflux chemotherapeutic agents (Cole et al., 1994; Cole, 2014a). MRP1 also protects normal tissues from xenobiotics when expressed at blood-tissue interfaces (Wijnholds et al., 1998). In addition, MRP1 mediates the efflux of many organic anions, including glutathione (GSH) and glucuronide-conjugated metabolites (Cole, 2014a, b). The physiologic relevance of MRP1 is associated with its ability to efflux GSH, as well as cysteinyl leukotriene (CysLT) C4 (LTC4) and 17β-estradiol 17-(β-d-glucuronide) (E217βG) (Jedlitschky et al., 1996; Loe et al., 1996).

The substrate specificities of MRP2/ABCC2 and MRP1 are similar, but unlike MRP1, MRP2 is found predominantly in the liver, where it helps maintain biliary homeostasis through elimination of conjugated metabolites (Nies and Keppler, 2007). In addition, MRP2 deficiency is the underlying cause of Dubin-Johnson syndrome, a disorder characterized by conjugated hyperbilirubinemia (Kartenbeck et al., 1996).

MRP3/ABCC3 is most closely related to MRP1, but like MRP2 it has a narrower tissue distribution and substrate specificity. Unlike MRP1 and MRP2, MRP3 does not transport GSH and only poorly transports LTC4 and other GSH conjugates although it shares an ability to transport E217βG (Kool et al., 1999). Hepatic MRP3 has a role in the disposition of certain drugs and/or their metabolites (van de Wetering et al., 2007).

The substrate specificity of the short MRP4/ABCC4 overlaps to a lesser extent with the long MRP1–3. Thus MRP4 is involved in the tissue distribution and elimination of antimetabolites used to treat viral and neoplastic diseases (Schuetz et al., 1999; Slot et al., 2011; Park et al., 2014). It is also distinct in its ability to efflux prostanoids [e.g., prostaglandin E2 (PGE2)] and cyclic nucleotides (Reid et al., 2003; Lin et al., 2008; Jin et al., 2014). However, like MRP1–3, MRP4 can transport E217βG.

There has been a long-standing interest in identifying small-molecule inhibitors of MRP1-mediated drug efflux in human tumors (Boumendjel et al., 2005; Cole, 2014a). This interest extended to other MRPs as their roles in drug distribution and elimination have been elucidated. MK-571 was originally developed as a CysLT receptor (CysLTR) antagonist to treat asthma (Jones et al., 1989). However, it also sensitizes tumor cells expressing MRP1, and inhibits MRP1-mediated transport of organic anions including LTC4 (Gekeler et al., 1995; Cole, 2014a). MK-571 also modulates transport by other MRPs (Keppler, 2011), other ABC transporters (Matsson et al., 2009), the solute carrier OATP1B3/SLCO1B3 (Letschert et al., 2005) and flavonol conjugation (Barrington et al., 2015). Despite its well documented lack of selectivity, MK-571 is still the most widely used small-molecule MRP inhibitor today.

The CysLTs LTC4 and LTD4 act on two classes of G protein-coupled CysLTRs (Haeggström and Funk, 2011). CysLT1R has a prominent role in the pathogenesis of asthma, and is selectively targeted by the experimental agents MK-571 and LY171883, and the clinically used montelukast and pranlukast (Fig. 1A) (Lynch et al., 1999; Sarau et al., 1999) . The role of CysLT2R in human health and disease is less well understood. It is found in immune cells and the vasculature of several tissues, including brain and heart (Heise et al., 2000; Nothacker et al., 2000). Two experimental CysLT2R-selective inhibitors are available, HAMI 3379 and BayCysLT2 (Fig. 1B) (Wunder et al., 2010; Ni et al., 2011).

Fig. 1.
Fig. 1.

Chemical structures of the LTMs investigated for their MRP modulatory properties in this study. The LTMs shown are selective antagonists of (A) CysLT1R, (B) CysLT2R, or (C) both (CysLT1/2R).

The development of antagonists or leukotriene modifiers (LTMs) that can distinguish between CysLT1R and CysLT2R raises the possibility that, unlike MK-571, one or more of them might also be selective for one of the MRP homologs. The goal of the present study was to test this idea by comparing the effects of seven CysLT1R-selective, CysLT2R-selective, and non-CysLTR-selective LTMs on organic anion transport by MRP1–4.

Materials and Methods

Materials.

AMP, ATP, and E217βG were from Sigma-Aldrich (Oakville, ON, Canada). LTC4 was from Calbiochem (San Diego, CA). Dulbecco’s modified Eagle’s medium (DMEM), OptiMEM, and Lipofectamine 2000 were from Gibco/Life Technologies (Burlington, ON) and protease inhibitors were from Roche (Mississauga, ON). MK-571 ((E)-​3-​[[[3-​[2-​(7-​chloro-​2-​quinolinyl)ethenyl]phenyl][[3-​(dimethylamino)-​3-​oxopropyl]thio]methyl]thio]-​propanoic acid) (Fig. 1A), montelukast (1-​[[[(1R)-​1-​[3-​(1E)-​2-​(7-​chloro-​2-​quinolinyl)ethenyl]phenyl]-​3-​[2-​(1-​hydroxy-​1-​methylethyl)phenyl]propyl]thio]-​methyl]-​cyclopropaneacetic acid)) (Fig. 1A), pranlukast (N-​[4-​oxo-​2-​(1H-​tetrazol-​5-​yl)-​4H-​1-​benzopyran-​8-​yl]-​4-​(4-​phenylbutoxy)-​benzamide) (Fig. 1A), LY171883 (1-​[2-​hydroxy-​3-​propyl-​4-​[4-​(1H-​tetrazol-​5-​yl)butoxy]phenyl]-​ethanone) (Fig. 1A), BAY-u9773 (4-​[[(1R,​2E,​4E,​6Z,​9Z)-​1-​[(1S)-​4-​carboxy-​1-​hydroxybutyl]-​2,​4,​6,​9-​pentadecatetraenyl]thio]-​benzoic acid) (Fig. 1C), and PGE2 were from Cayman Chemical (Ann Arbor, MI). HAMI 3379 (3-​[[(3-​carboxycyclohexyl)amino]carbonyl]-​4-​[3-​[4-​[4-​(cyclohexyloxy)butoxy]phenyl]propoxy]-​benzoic acid) (Fig. 1B) and BayCysLT2 (3-​[[(3-​carboxycyclohexyl)amino]carbonyl]-​4-​[3-​[4-​(4-​phenoxybutoxy)phenyl]propoxy]-​benzoic acid) (Fig. 1B) were kind gifts from Dr. Colin Funk (Queen’s University, Kingston, ON). [6,7-3H(N)]E217βG (42 Ci mmol−1), [14,15,19,20-3H(N)]LTC4 (166.3 Ci mmol−1), and [5,6,8,11,12,14,15-3H(N)]PGE2 (153.7 Ci mmol−1) were from PerkinElmer Life Sciences (Boston, MA). Murine monoclonal antibody (mAb) M2Ι-4 (anti-MRP2) (ALX-801-015) and rat mAb M4Ι-10 (anti-MRP4) (ALX-801-038) were from Enzo (Farmingdale, NY). Rabbit anti-Na+/K+-ATPase α (H-300) (SC-28800) and murine mAb M3ΙΙ-9 (anti-MRP3) (SC-59613) were from Santa Cruz Biotechnology (Dallas, TX). The human MRP1-specific mAb QCRL-1 was generated in this laboratory (Hipfner et al., 1994). Horseradish peroxidase (HRPase)-conjugated goat anti-mouse antibody (BML-SA204) was from Pierce Biotechnology (Rockford, IL), HRPase-conjugated goat anti-rabbit antibody (CED-CLAS10-667) was from Cedarlane (Burlington, ON), and HRPase-conjugated goat anti-rat antibody (AP136P) was from EMD Millipore (Billerica, MA). Western Lightning Plus-enhanced chemiluminescence blotting reagents were from PerkinElmer.

Cell Culture and Transfection.

The stably transfected MRP1-expressing human embryonic kidney (HEK) 293 cell line has been previously described (Myette et al., 2013). The stably transfected MRP2- and MRP3-expressing HEK293 cell lines were established using pCEBV7-based expression vectors generated by moving the MRP coding sequences from pcDNA3.1(–)-MRP2 (Ito et al., 2001) and pcDNA3.1(+)-MRP3 (Oleschuk et al., 2003) into a modified pCEBV7 vector as described (Cole et al., 1994). HEK293T cells transiently expressing human MRP4 were generated using pcDNA3.1(–)-MRP4 and Lipofectamine 2000 as described (Myette et al., 2013). All cell lines were grown in DMEM/7.5% fetal bovine serum at 37°C in 5% CO2/95% air. After collection, cells were overlaid with homogenization buffer (250 mM sucrose/50 mM Tris pH 7.4/0.25 mM CaCl2) with protease inhibitors, and cell pellets stored at –80°C until needed.

Preparation of MRP-Enriched Membrane Vesicles and Immunoblotting.

Membrane vesicles were prepared from MRP-transfected HEK cells as previously described (Loe et al., 1996). Membrane vesicle protein was quantified using a Bradford assay and the presence of MRP1, MRP2, MRP3, and MRP4 in the membrane vesicles was confirmed by immunoblotting as before (Létourneau et al., 2007; Myette et al., 2013). Blots were incubated overnight at 4°C with the appropriate MRP-specific antibodies as well as with anti-Na+/K+-ATPase as a membrane-protein-loading control. Bound antibodies were detected using HRPase-conjugated secondary antibodies followed by chemiluminescence blotting reagents and exposure of blots to X-ray film.

ATP-Dependent MRP-Mediated Uptake of 3H-Labeled Organic Anions into Inside-Out Membrane Vesicles.

The ATP-dependent uptake of 3H-labeled E217βG, LTC4, and PGE2 into inside-out vesicles was measured using a modified rapid filtration method adapted to a 96-well plate format (Loe et al., 1996; Létourneau et al., 2007). Stock solutions of LTMs prepared in either dimethyl sulfoxide or ethanol were diluted so that the final vehicle concentration in the reaction mixture was <1%. Transport reactions were carried out in a final volume of 30 μl consisting of 50 mM Tris/250 mM sucrose buffer (TSB), pH 7.4, 4 mM ATP (or AMP), 10 mM MgCl2, the desired concentration of LTM, membrane vesicle protein, and 3H-labeled substrate as follows: MRP1, 2 μg protein, [3H]E217βG (400 nM, 20 nCi) or [3H]LTC4 (50 nM, 10 nCi); MRP2, 4 μg protein, [3H]E217βG (400 nM, 40 nCi); MRP3, 4 μg protein, [3H]E217βG (1 μM, 80 nCi); MRP4, 5 μg protein, [3H]E217βG (30 μM, 60 nCi), or [3H]PGE2 (5 μM, 100 nCi) (Létourneau et al., 2007; Myette et al., 2013). Assays were carried out in duplicate at 37°C for [3H]E217βG and [3H]PGE2 uptake and at 23°C for [3H]LTC4 uptake (Loe et al., 1996). The optimal (linear) uptake times for each MRP and organic anion substrate were determined in preliminary experiments to be as follows: MRP1, E217βG, 3 minutes; MRP2, E217βG, 7 minutes; MRP3, E217βG, 7 minutes; MRP4, E217βG, 10 minutes; MRP1, LTC4, 1 minutes; MRP4, PGE2, 10 minutes. Uptake was stopped by dilution in ice-cold TSB and the reaction contents filtered onto a Unifilter-96 GF/B filter plate (Perkin-Elmer) using a FilterMate Harvester apparatus (Packard BioScience Company, Meriden, CT). Tritium associated with the vesicles was quantified using a TopCount NXT Microplate Scintillation and Luminescence Counter (Perkin-Elmer). ATP-dependent uptake of 3H-labeled organic anions was calculated by subtracting uptake in the presence of AMP from uptake in the presence of ATP; results are expressed as a percent of uptake in the absence of the LTM.

Half-maximal inhibitory (IC50) and half-maximal stimulatory (SC50) values were determined by curve-fitting with nonlinear regression using GraphPad Prism 6.0 software (GraphPad, San Diego, CA). Experiments were repeated two or more times using at least two independent membrane vesicle preparations, and the mean SC50 and/or IC50 values (± S.D.) determined. One-way analysis of variance with a Tukey’s multiple comparisons posthoc test was performed using GraphPad Prism 6.0; P values < 0.05 were considered significant.

Results

All LTMs Inhibit Vesicular [3H]E217βG Transport by MRP1.

To compare the relative abilities of the seven LTMs depicted in Fig. 1 to modulate MRP1-mediated transport, vesicular uptake assays using the common MRP substrate [3H]E217βG were performed using MRP1-enriched membrane vesicles prepared from stably transfected HEK cells. The presence of MRP1 in the membrane vesicles was first confirmed by immunoblot analysis, and as expected, a single band at 190 kDa corresponding to MRP1 was observed (Fig. 2A).

Fig. 2.
Fig. 2.

Immunoblots of MRP-enriched membrane vesicles. Levels of MRP1, MRP2, MRP3, and MRP4 proteins were detected in membrane vesicles prepared from transfected HEK293 cells by immunoblotting. Membrane vesicles derived from untransfected HEK293 cells were used as a control. (A) MRP1, (B) MRP2, (C) MRP3, and (D) MRP4 were detected with mAbs QCRL-1, M2I-4, M3II-9, and M4I-10, respectively; anti-Na+/K+-ATPase was used as a protein loading control.

The IC50 values of the seven LTMs were then determined by measuring their effects on E217βG uptake at 6–7 LTM concentrations (range 0.01–40 μM) and sigmoid curves fitted to the datasets. Representative concentration-response curves are shown in Fig. 3 and the mean IC50 values obtained from multiple independent experiments are summarized in Table 1. For MRP1-mediated E217βG uptake, the IC50s of the LTMs ranged from 1.6 to 16.1 μM and no stimulation was observed at any of the concentrations tested. The IC50s (1.8–16.1 μM) of the four CysLT1R-selective LTMs (MK-571, montelukast, pranlukast, LY171883) (Fig. 1A) were generally greater and broader than those of the CysLT2R-selective HAMI 3379 and BayCysLT2 (Fig. 1B) (IC50s 1.6 and 1.9 μM, respectively). However, the IC50s of the most potent CysLT1R-selective LTMs (MK-571 and pranlukast, 1.8 and 2.8 μM, respectively) were comparable to the IC50s of the CysLT2R-selective LTMs and the nonselective BAY-u9773 (Fig. 1C) (1.6–1.9 μM) (P > 0.05) (Table 1). Overall, the data indicate that the CysLTR selectivity of the LTMs does not correlate with their potencies as inhibitors of MRP1-mediated E217βG uptake.

Fig. 3.
Fig. 3.

The inhibitory effects of LTMs on E217βG transport by MRP1. Representative concentration-response curves illustrating the inhibitory effects of increasing LTM concentrations on ATP-dependent MRP1-mediated E217βG uptake into membrane vesicles: (A) MK-571, (B) montelukast, (C) pranlukast, (D) HAMI 3379, (E) BayCysLT2, and (F) BAY-u9773. Each data point is the mean of duplicate determinations in a single experiment. IC50 values derived from multiple independent experiments are summarized in Table 1 (LTM concentration range tested 0.01–40 μM).

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TABLE 1

Inhibition of MRP1-mediated vesicular uptake of E217βG and LTC4 by LTMs

LTMs Can Stimulate, Inhibit, or Have a Biphasic Effect on MRP2-Mediated Vesicular [3H]E217βG Transport.

To determine the ability of the LTMs to modulate MRP2-mediated E217βG transport, uptake assays were performed using MRP2-enriched membrane vesicles prepared from stably transfected HEK cells. The presence of MRP2 in the membrane vesicles was first confirmed by immunoblotting, and as expected a single band at 190 kDa corresponding to MRP2 was observed (Fig. 2B).

MRP2-mediated [3H]E217βG transport was then measured in the presence of the LTMs at 6–14 concentrations (range 0.1–175 μM). As shown in Fig. 4 (and summarized in Table 2), three of four CysLT1R-selective LTMs (MK-571, montelukast, and pranlukast) had biphasic effects on E217βG transport with stimulation of uptake by MRP2 at lower LTM concentrations (1 to ∼10 μM), and inhibition of uptake at higher concentrations (Fig. 4, A–C). The maximal stimulation of E217βG uptake observed ranged from 1.3- to 1.6-fold (at 2–6 μM LTM).

Fig. 4.
Fig. 4.

The inhibitory, stimulatory, and biphasic modulatory effects of LTMs on E217βG transport by MRP2. Representative concentration-response curves illustrating the modulatory effects of increasing LTM concentrations on MRP2-dependent E217βG uptake into membrane vesicles: (A) MK-571, (B) montelukast, (C) pranlukast, (D) LY171883, (E) HAMI 3379, (F) BayCysLT2, and (G) BAY-u9773. Biphasic modulatory effects (A and B) are defined as stimulation of E217βG uptake by MRP2 at lower LTM concentrations followed by an inhibition of E217βG uptake at higher concentrations. Each data point is the mean of duplicate determinations in a single experiment. IC50 and SC50 values derived from multiple independent experiments are summarized in Table 2 (LTM concentration range tested 0.03–175 μM).

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TABLE 2

Modulation of MRP2- and MRP3-mediated vesicular uptake of E217βG by LTMs

The fourth CysLT1R-selective LTM, LY171883, only stimulated MRP2-mediated E217βG transport (maximal 4-fold stimulation at 100 μM, 3.2-fold at 30 μM) (Fig. 4D; Table 2). This contrasts with its effect on MRP1 transport activity, where only inhibition was observed over the same concentration range (Table 1). The SC50 value of LY171883 was 11.3 μM, which was significantly higher than the SC50s for MK-571, montelukast, and pranlukast (range 1.0–2.6 μM) (P < 0.05) (Table 2).

As inhibitors of MRP2 activity, MK-571, montelukast, and pranlukast were 2- to 6-fold less potent (IC50s 21.2, 26.9, and 23.7 μM, respectively) than the CysLT2R-selective and nonselective LTMs (P < 0.05) (Fig. 4, A–C, versus Fig. 4, E–G; Table 2). On the other hand, the IC50s for the CysLT2R-selective HAMI 3379 and BayCysLT2 and the nonselective BAY-u9773 were comparable to each other at 4.3 μM, 12.2 μM, and 8.0 μM, respectively (P > 0.05) (Fig. 4, E–G; Table 2).

Together these data indicate that CysLTR selectivity of the LTMs may weakly correlate with their ability to modulate MRP2 transport activity since 1) stimulation of E217βG uptake was only observed in the presence of CysLT1R-selective LTMs; and 2) when inhibition of E217βG uptake was observed, the CysLT1R-selective LTMs (with the exception of LY171883) were significantly less potent inhibitors (up to 6-fold) than the CysLT2R-selective and nonselective LTMs.

LTMs Can Both Inhibit and Stimulate [3H]E217βG Uptake by MRP3 but Do Not Elicit a Biphasic Response.

To further explore their possible MRP selectivity, the effects of the LTMs on E217βG uptake by MRP3 were determined using MRP3-enriched membrane vesicles prepared from stably transfected HEK cells. MRP3 levels were first confirmed by immunoblotting as before (Fig. 2C). Two closely migrating immunoreactive bands at ∼190 kDa corresponding to the molecular weight of MRP3 were observed and are presumed to represent variably glycosylated forms of the transporter.

The LTMs were tested at six concentrations (range 0.1–175 μM), and in all instances with one exception, they inhibited MRP3-mediated E217βG uptake and no stimulation was observed (Fig. 5A, B; D–F; Table 2). The exception was LY171883, which only stimulated E217βG uptake by MRP3 at concentrations up to 30 μM (Fig. 5C; Table 2), as it did for MRP2-mediated E217βG uptake (Fig. 4C; Table 2). Thus, in contrast to MRP2 and when compared over the same concentration ranges, none of the LTMs had a biphasic effect on MRP3 transport.

Fig. 5.
Fig. 5.

The inhibitory and stimulatory effects of LTMs on E217βG transport by MRP3. Representative concentration-response curves illustrating the modulatory effects of increasing LTM concentrations on MRP3-dependent E217βG uptake into membrane vesicles: (A) montelukast, (B) pranlukast, (C) LY171883, (D) HAMI 3379, (E) BayCysLT2, and (F) BAY-u9773. Each data point is the mean of duplicate determinations in a single experiment. IC50 and SC50 values derived from multiple independent experiments are summarized in Table 2 (LTM concentration range tested was 0.1–175 μM).

The CysLT1R-selective montelukast and pranlukast inhibited MRP3-mediated E217βG uptake with comparable IC50s of 20.3 μM and 19.2 μM (P > 0.05), respectively, whereas MK-571 inhibited uptake by 57.0 ± 4.0% at 30 μM and by 96.3 ± 0.2% at 100 μM (Fig. 5, A and B; Table 2). The CysLT2R-selective HAMI 3379 and BayCysLT2, and the nonselective BAY-u9773 were more potent inhibitors (∼2.6- to 4.9-fold) with IC50s of 4.3 μM, 7.7 μM, and 4.0 μM, respectively (P < 0.05) (Fig. 5, D–F; Table 2). In contrast, the CysLT1R-selective LY171883 stimulated MRP3-mediated E217βG transport by a maximum of approximately 2-fold at 30 μM (SC50 8.1 μM) (Fig. 5C; Table 2), which was similar to observations for MRP2 (SC50 11.3 μM). Thus, as shown for MRP2 (but again with the exception of LY171883), the CysLT1R-selective LTMs were less potent inhibitors of MRP3 transport activity (up to 5-fold) than the CysLT2R-selective and nonselective LTMs.

All LTMs Can Inhibit MRP4-Mediated [3H]E217βG Uptake.

Because the LTMs had differential effects on E217βG uptake by the long MRP1–3, it was of interest to determine their effects on E217βG uptake by the more distantly related short MRP4. Thus, vesicular uptake assays were performed using membrane vesicles prepared from transfected HEK cells enriched for MRP4 as demonstrated by immunoblotting (Fig. 2D). As reported earlier, two immunoreactive bands close to the expected molecular mass of MRP4 (170 kDa) were detected and are presumed to be variably glycosylated forms of the transporter (Myette et al., 2013).

The seven LTMs were tested at six concentrations (range 0.1–100 μM) for their ability to modulate MRP4-mediated [3H]E217βG uptake, and inhibition was observed in all cases (Fig. 6, A–G). Thus, in contrast to MRP2 and MRP3, but like MRP1, none of the seven LTMs stimulated MRP4-mediated E217βG uptake at any of the concentrations tested. Mean IC50 values from multiple independent experiments are summarized in Table 3.

Fig. 6.
Fig. 6.

The inhibitory effects of LTMs on E217βG transport by MRP4. Shown are representative concentration-response curves illustrating the inhibitory effects of increasing LTM concentrations on MRP4-dependent E217βG uptake into membrane vesicles. (A) MK-571, (B) montelukast, (C) pranlukast, (D) LY171883, (E) HAMI 3379, (F) BayCysLT2, and (G) BAY-u9773. Each data point is the mean of duplicate determinations in a single experiment. IC50 values derived from multiple independent experiments are summarized in Table 3 (LTM concentration range tested 0.1–100 μM).

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TABLE 3

Inhibition of MRP4-mediated vesicular uptake of E217βG and PGE2 by LTMs

As with MRP1, the IC50 values for the CysLT1R-selective LTMs (montelukast, pranlukast, LY171883) (3.3–18.5 μM) were generally greater and broader than the IC50s for the CysLT2R-selective HAMI 3379 and BayCysLT2 (1.2 and 1.9 μM, respectively). However, the IC50s of the most potent CysLT1R-selective LTMs (pranlukast, 3.3 μM; MK-571, 2.2 μM) were comparable to the IC50s of the CysLT2R-selective LTMs (1.2 and 1.9 μM) as well as the nonselective BAY-u9773 (2.6 μM) (Table 3) (P > 0.05). Thus, these data indicate that, as observed for MRP1 (Table 1), the CysLTR selectivity of the LTMs does not correlate with their potency as inhibitors of MRP4-mediated E217βG transport.

Analysis of the MRP Selectivity of the LTMs.

To better illustrate the effects of an individual LTM on MRP1–4 transport activities, the IC50s for the [3H]E217βG uptake data as summarized in Tables 1–3 were replotted against the four different MRP homologs (Supplemental Fig. 1). When the data are presented in this fashion, it becomes more apparent that the CysLT1R-selective LTMs are generally less potent than the CysLT2R-selective and dual-selective BAY-u9773 LTMs, and that IC50 values for inhibition of E217βG transport by MRP1 and MRP4 are closer than those for transport by MRP2 and MRP3.

LTMs Do Not Modulate MRP1- and MRP4-Mediated Organic Anion Transport in a Substrate-Specific Manner.

To compare the ability of the LTMs to inhibit E217βG transport with their ability to inhibit the transport of a more MRP homolog–specific organic anion substrate, MRP1-mediated uptake of its physiologic and highest affinity substrate LTC4 was measured in the presence of LTMs at 10 concentrations (range 0.01–175 μM). IC50s were determined as before and the results are summarized in Table 1. All seven LTMs inhibited LTC4 uptake as they did MRP1-mediated E217βG uptake, with mean IC50 values ranging from 2.7 to 52.2 μM (for E217βG transport, the range was 1.6–16.1 μM). The general rank order potencies of inhibition by the LTMs for LTC4 and E217βG uptake by MRP1 were also similar (Table 1). These data indicate that the LTMs do not differentially affect the transport of these two organic anion substrates of MRP1.

The seven LTMs were also tested for their ability to modulate uptake of [3H]PGE2 by MRP4, a physiologic substrate of this short MRP that is not transported by MRP1, 2, or 3 (Reid et al., 2003; Lin et al., 2008). Thus PGE2 uptake into MRP4-enriched membrane vesicles was measured in the presence of the LTMs at six concentrations (range 0.1–100 μM) and IC50s determined as before. As summarized in Table 3, all seven LTMs inhibited PGE2 uptake as they did MRP4-mediated E217βG uptake (Fig. 6, Table 3). The mean IC50s for PGE2 transport ranged from 1.1 to 9.9 μM (for MRP4-mediated E217βG transport, the range was 1.2–18.5 μM). The rank order of LTM inhibitory potencies for PGE2 uptake and E217βG uptake by MRP4 were also essentially the same, with the three CysLT2R-selective and nonselective LTMs tending to be more potent inhibitors than the four CysLT1R-selective LTMs (Table 3). These data indicate that the LTMs do not distinguish between at least these two organic anion substrates of MRP4.

Discussion

CysLT1R is the primary molecular target of MK-571 which blocks the LTD4-mediated activation of this receptor at low nanomolar concentrations (Lynch et al., 1999; Heise et al., 2000), whereas low-to-mid micromolar MK-571 concentrations are needed to inhibit transport by MRP1/ABCC1. Regardless of these differences in potency, the ability of MK-571 to inhibit MRP1 (and other transporters) is somewhat unexpected given the lack of sequence homology and structural similarity between CysLT1R and the MRPs. The data presented here and elsewhere show that the sensitivity of MRP1 and its homologs to modulation by MK-571 varies substantially when E217βG is used as a probe for transport activity (Tables 1–3). Thus, E217βG uptake by MRP3/ABCC3 appears to be least sensitive to inhibition by MK-571, and MRP1 and MRP4/ABCC4 most sensitive, with MRP2/ABCC2 in between. MK-571 is most often used with the intent of fully inhibiting MRP activity, and for this purpose our data indicate that the MK-571 concentrations needed to achieve >80% inhibition are 5 μM for MRP1, 30 μM for MRP2, 100 μM for MRP3, and 5 μM for MRP4. In intact cell or whole organism systems in which modulators must be taken up into the cell to exert their effects, the concentrations required to achieve MRP1–4 inhibition can be expected to be significantly higher. However, if 5 μM MK-571 is used to inhibit MRP1 in a vesicular transport system where the other homologs are present, MRP4 would also be inhibited, but MRP2 transport activity would be stimulated (see below).

Newer CysLT1R-selective LTMs also inhibit the transport of various MRP1 substrates but far less is known about their abilities to inhibit other MRP homologs (Nagayama et al., 1998; van Brussel et al., 2004; Conseil and Cole, 2013). To address this deficiency we investigated the effects of three additional CysLT1R-selective LTMs on the MRP1–4-mediated transport of E217βG. The patterns of inhibition of MRP1 and MRP4 by the antiasthmatic agents pranlukast and montelukast were comparable to the pattern observed for MK-571, whereas LY171883 was less potent (Tables 1 and 3). The responses of MRP2 and MRP3 to the CysLT1R-selective LTMs were more complex. Thus, MRP2 was inhibited by MK-571, montelukast, and pranlukast only at higher concentrations, but at lower concentrations (1–∼10 μM), MRP2-mediated E217βG uptake was stimulated (up to 1.6-fold). In contrast, these same three CysLT1R-selective LTMs exerted only inhibitory effects on MRP3. However, this was not the case for LY171883, a CysLT1R antagonist that can also act as a phosphodiesterase inhibitor (Fleisch et al., 1985) and a peroxisome-proliferating agent (Foxworthy et al., 1990). Thus, LY171883 only stimulated E217βG uptake by MRP2 and MRP3 (up to 4-fold and 2-fold, respectively) and did not exert the biphasic actions of the other CysLT1R-selective LTMs, at least at the concentrations tested, which include those likely to be used in vesicular transport studies in vitro.

The comparable sensitivities of MRP1 and MRP4 to the CysLT1R-selective LTMs is of interest because of the substantial structural and substrate specificity differences between the long MRP1 and short MRP4. The sequence similarity between MRP1 and MRP4 is just 31%, whereas the sequence similarities among the long MRP1–3 are significantly greater (46–56%). The chemical properties of the solutes transported by MRP4 are also more distinct than those transported by MRP1–3, and the few substrates that MRP1 and MRP4 do have in common (e.g., E217βG) differ considerably in their transport kinetics (Km(app) 1.7 versus 17 μM, respectively) (Jedlitschky et al., 1996; Wittgen et al., 2012). It seems improbable that MRP1 and MRP4 have a common set of contact amino acids for these LTMs that enables them to inhibit transport of E217βG (as well as their more homolog-specific substrates LTC4 and PGE2, respectively) with comparable potency. A better explanation may be more forthcoming when more detailed structural information on the MRPs becomes available.

The biphasic modulatory effects observed with the CysLT1R-selective MK-571, montelukast, and pranlukast on E217βG uptake by MRP2 and the stimulatory effects observed with LY171883 on E217βG uptake by both MRP2 and MRP3 are reminiscent of earlier reports on these transporters from our group and others. Thus a structurally diverse array of compounds, including cannabinoid type 1 (CB1) receptor antagonists (e.g., rimonabant), ethinyl estradiol conjugates, sulfinpyrazone, indomethacin, and chalcogenopyrylium dyes have been reported to modulate organic anion transport by MRP2 (and MRP3) in a complex fashion (Bakos et al., 2000; Bodo et al., 2003; Zelcer et al., 2003; Chu et al., 2004; Gerk et al., 2004; Wittgen et al., 2011; Myette et al., 2013). To explain the biphasic response to some modulators, it has been proposed that the transporter contains both a transport site and an allosteric modulatory site whereby the modulator stimulates the transporter allosterically at low probe (E217βG) concentrations and competes for the E217βG binding site at higher concentrations (Zelcer et al., 2003; Bodo et al., 2003). The observation that LY171883 only stimulates E217βG transport by MRP2 and MRP3 suggests that this LTM can occupy only the allosteric site on these transporters. MRP4 is also thought to contain multiple allosteric substrate binding sites (Van Aubel et al., 2005), but we saw no evidence of an allosteric interaction of the LTMs with the E217βG or PGE2 probes used here. Whether the CysLT1R-selective LTMs (and the CysLT2R-selective LTMs discussed below) are themselves actively transported by the MRPs or simply occupy a substrate or allosteric modulatory binding site remains to be determined.

Drug-drug interactions have been implicated with the concomitant use of montelukast and an array of other drugs, including efavirenz and ivacaftor, and have been attributed to alterations in oxidative metabolism (Ibarra-Barrueta et al., 2014; Schneider et al., 2015). However, the metabolites of montelukast and pranlukast are excreted primarily via the hepatobiliary elimination route, where modulation of MRP2 activity may have pharmacokinetic consequences (Keam et al., 2003; Diamant et al., 2009). Thus, our observations with the CysLT1R-selective LTMs suggest a potential role for the MRPs in drug-drug interactions in patients taking this class of antiasthma medication. Further studies using intact cell assays and appropriate Abcc knockout mice may be helpful in evaluating this possibility.

The CysLT2R-selective LTMs have only recently become available, but their usefulness in elucidating the role of CysLT2R in multiple cellular processes including vascular permeability and ischemic injury is already well established (Ni et al., 2011; Zhang et al., 2013). HAMI 3379 is reported to be 10,000-fold more selective and BayCysLT2 500-fold more selective for CysLT2R than CysLT1R (Wunder et al., 2010; Ni et al., 2011). However, until now, the (possible) effects of the CysLT2R-selective LTMs on the transport activities of the MRPs have not been investigated. Our data show that, despite their ability to distinguish between the two CysLTR isoforms, HAMI 3379 and BayCysLT2 show little or no such selectivity with respect to inhibiting E217βG transport by the four MRP homologs. However, like the CysLT1R-selective LTMs, the two CysLT2R-selective LTMs more potently inhibited E217βG transport by MRP1 and MRP4 (IC50s < 2 μM) than by MRP2 and MRP3 (IC50s < 12 μM). In contrast, unlike the CysLT1R-selective LTMs, neither HAMI 3379 nor BayCysLT2 stimulated or caused a biphasic effect on MRP2 and MRP3 transport activity. Finally, as an MRP modulator, the nonselective or dual CysLTR antagonist BAY-u9773 behaved much more like HAMI 3379 and BayCysLT2 than it did the CysLT1R-selective LTMs, despite the fact that BAY-u9773 is 20- to 500-fold less potent at blocking LTC4-mediated CysLT2R activation (Ni et al., 2011).

The data presented here and elsewhere highlight the ongoing need for more selective small-molecule inhibitors of the individual MRP homologs. The tricyclic isoxazole LY475776 and ceefourin-2 have been identified as relatively selective inhibitors of MRP1 and MRP4, respectively (Dantzig et al., 2004; Cheung et al., 2014), but to our knowledge no MRP2 or MRP3 selective inhibitors have been reported. However, it seems evident that both existing and newly developed MRP inhibitors require extensive testing for activity against a broad range of both efflux and import transporters in a variety of intact cell and cell-free assay systems to substantiate all claims of MRP homolog selectivity.

In conclusion, the comparative studies reported here clearly illustrate the nonselectivity of both classes of CysLTR antagonists with respect to their ability to modulate organic anion transport by MRP1–4, and the consequent limitations of these LTMs as small-molecule inhibitors in studies of these transporters. Particularly with respect to MRP2 and MRP3, the possible opposing and concentration-dependent effects of the CysLT1R-selective LTMs have the potential to confound interpretation of observations in systems (e.g., cell culture and intact animal models) in which multiple MRP homologs (and non-ABC transporters and conjugating systems) are probably present. On the other hand, our studies are the first to show that CysLT2R-selective HAMI 3379 and BayCysLT2 are also potent inhibitors of MRP1–4-mediated transport (e.g., HAMI 3379 IC50s 1.2–4.3 μM). Unlike the more commonly used CysLT1R-selective LTMs, these CysLT2R-selective antagonists did not stimulate transport by any of MRP1–4 at comparable concentrations. As such, they may be attractive alternatives to the CysLT1R-selective LTMs as pharmacological tools in MRP studies until better homolog-specific inhibitors become available.

Acknowledgments

The authors wish to thank Drs. Colin Funk and James F. Brien of Queen’s University for helpful discussions, and Kathy Sparks for tissue culture assistance.

Authorship Contributions

Participated in research design: Csandl, Conseil, Cole.

Conducted experiments: Csandl, Conseil.

Performed data analysis: Csandl, Conseil, Cole.

Wrote or contributed to the writing of the manuscript: Csandl, Conseil, Cole.

Footnotes

    • Received January 13, 2016.
    • Accepted April 7, 2016.

  • This work was supported by the Canadian Institutes of Health Research [MOP-106513, MOP-133584]. M.A.C. was the recipient of the Eldon Boyd Fellowship from Queen’s University. S.P.C.C. is Canada Research Chair in Cancer Biology and Bracken Chair in Genetics and Molecular Medicine.

  • dx.doi.org/ 10.1124/dmd.116.069468.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

ABC
ATP-binding cassette
CysLT
cysteinyl leukotriene
CysLTR
CysLT receptor
E217βG
17β-estradiol 17-(β-d-glucuronide)
GSH
glutathione
HEK
human embryonic kidney
HRPase
horseradish peroxidase
LTC4
leukotriene C4
LTM
leukotriene modifier
mAb
monoclonal antibody
MRP
multidrug resistance protein
PGE2
prostaglandin E2

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Transport by MRP1–4 Modulated by CysLT Receptor Antagonists

Mark A. Csandl, Gwenaëlle Conseil and Susan P. C. Cole
Drug Metabolism and Disposition June 1, 2016, 44 (6) 857-866; DOI: https://doi.org/10.1124/dmd.116.069468
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