drug metabolism and transport || multidrug resistance proteins and hepatic transport of endo- and...

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Multidrug Resistance Proteins and HepaticTransport of Endo- and Xenobiotics

Phillip M. Gerk and Mary Vore

SummaryTransporters that use ATP are called ATP-binding cassette (ABC) transporters. One family of

ABC transporters that plays a major role in the ability of the liver to eliminate various drugs isthe multidrug resistance proteins (MRPs). There are nine cloned genes in the MRP (ABCC) sub-family. Generally, these carriers transport diverse sulfate, glutathione, or glucuronide conjugatesof endogenous compounds like estrogen and bilirubin, as well as drugs and toxins. Although allof the MRPs mediate ATP-dependent efflux, their tissue expression and cellular localization vary.MRP2 is most highly expressed in the liver at the canalicular domain and is the primary focusof this chapter. Several in vitro approaches to study drug transport in the liver are discussed.Although these approaches have been described in detail elsewhere, the authors present theirown perspectives, with particular emphasis on the baculovirus expression vector system. Addi-tional methods and approaches that are discussed include the choice of animal tissue preparation,expression system to be used, and experimental paradigms, including uptake in membrane vesi-cles, cell monolayers, and procedures to measure ATPase activity and its relationship to transport.

Key WordsATPase activity; ATP-binding cassette transporters; baculovirus expression; expression

systems; hepatocytes; liver; membrane vesicles; multidrug resistance associated proteins; Sf9cell culture, Sf9 membrane vesicle preparation.

1. INTRODUCTIONMost biologically active compounds can be described in terms of their

degree of lipophilicity or hydrophilicity. Lipophilic compounds can passthrough lipid membranes rapidly, so movement is limited by aqueous diffu-sion. Hydrophilic compounds do not pass through membranes easily, and thus

From: Methods in Pharmacology and Toxicology,Drug Metabolism and Transport: Molecular Methods and Mechanisms

Edited by: L. Lash © Humana Press Inc., Totowa, NJ

require a transporter to facilitate their movement into and out of cells (1). Ofcourse, many compounds fall in between these extremes, and the need forprotein-mediated transport is determined by the mass movement required toobtain or avoid their physiologic, pharmacologic, or toxicologic activities, asthe case may be. This review focuses on protein-mediated transport processesrather than simple diffusion.

Protein-mediated transport is accomplished by channels and transporters.Channels serve as gated pores that regulate the rapid movement of ions acrossmembranes, and are not further discussed in this chapter. Transporters fall intoseveral categories, which are all saturable. Facilitated diffusion carriers do notrequire energy and simply accelerate the diffusion of their substrates down theirelectrochemical potential gradient (downhill transport). In contrast, active trans-porters can build and maintain concentration gradients (uphill transport). Pri-mary active transporters use nucleotides like ATP (or rarely, light) as an energysource, using the release of energy to drive the conformational change thattranslocates the transport substrate. Allocrite is another formal term used todescribe the transported compound (informally referred to as “substrate”), todistinguish it from ATP, which is a true substrate hydrolyzed by ATP-dependenttransporters (2). Secondary (and tertiary) active transporters use the change infree energy from the downhill transport of one compound to drive the uphilltransport of another. The general properties of transporters have been describedin greater detail by Stein (1,3).

Recently, transporters have been classified into two superfamilies. Trans-porters that use ATP are called ATP-binding cassette (ABC) transporters andhave been recently reviewed (4,5). All other transporters are classified in thesolute carrier (SLC) superfamily. More information on nomenclature, classifi-cation, and functions of transporters can be found at the websites in Table 1.

Clearly, the liver plays a major role in drug elimination, both through metab-olism and transport. Several important hepatic transporters are shown in Fig. 1(5,6). The main movement of solutes is from the basolateral side (where uptaketransporters predominate) to the canalicular (apical) side (where efflux trans-porters predominate). The solutes will then flow with bile through the bile ductsand eventually to the intestine. Thus, the liver has two related transport func-tions: bile formation and excretion. Bile formation results from the transport ofosmotically active solutes into the confined space of the canaliculus, followedby the passive movement of water until equilibrium is reached (7). Osmolytespresent in bile include bile acids, which are transported into the canaliculus bythe bile salt export pump (Bsep, Abcb11); the accompanying counterion, Na+,along with the bile acids contribute a major component of bile flow, termed bile

274 Gerk and Vore

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275

acid dependent bile flow (7). The significantly decreased bile flow in TR– rats,deficient in multidrug resistance associated protein 2 (Mrp2, Abcc2; discussedin greater detail below) provides significant support to the theory that Mrp2substrates, including glutathione and glucuronide conjugates, are also importantosmolytes in bile that are responsible for bile acid independent bile flow (8).Glutathione, an Mrp2 substrate, is considered to be the single most importantosmolyte contributing to bile acid independent bile flow (8). Thus, the transportfunctions of the liver are not independent of, but rather interactive with, metab-olism, as demonstrated with Mrp2 in the following subheading.

2. MRPS: FUNCTIONS AND MECHANISMSThere are nine cloned genes in the MRP (ABCC) subfamily. Generally, these

transporters transport diverse sulfate, glutathione, or glucuronide conjugates ofendogenous compounds like estrogen and bilirubin, as well as drugs and toxins(6,9). For example, MRP1-4 all transport β-estradiol-17-(β-D-glucuronide)(E217G), but with differing efficiencies (6). Although all of the MRPs mediateATP-dependent efflux, their tissue expression and cellular localization vary.MRP2 is most highly expressed in the liver at the canalicular domain, so thischapter focuses on MRP2. Genetic deficiency of MRP2 causes Dubin–Johnsonsyndrome, resulting in hyperbilirubinemia, decreased bile flow, decreased excre-tion of many endo- and xenobiotics, and deposition of a dark pigment in the

276 Gerk and Vore

Fig. 1. Basolateral and canalicular hepatic transporters. Basolateral transporters areshown on the left, canalicular transporters are shown on the right. BSEP, Bile saltexport pump; MDRs, multidrug resistance transporter (including human MDR1 andMDR3 and their rat homologues Mdr1a/1b and Mdr2); MRP, multidrug resistance-associated protein; MXR/BCRP, mitoxantrone resistance transporter/breast cancer resis-tance protein; NTCP, sodium-taurocholate cotransporting protein; OATP, organic aniontransporting polypeptides; OCT1, organic cation transporter 1; PGT, prostaglandintransporter.

liver. Like other MRPs, MRP2 mediates the excretion of conjugates (Fig. 2), butMRP2 shows less dependence on glutathione cotransport, compared to MRP1.

Mrp2 expression can be altered by pathologic states such as renal failure,pregnancy, cholestasis, and acute phase response (9). However, MRP3 isexpressed on the basolateral membrane, and its expression can be induced bycholestasis, serving as a potential protective mechanism against the intracellu-lar accumulation of toxic compounds and metabolites in the hepatocyte (11,12).In addition, farnesoid X receptor, pregnane X receptor, and constitutiveandrostane receptor ligands, and several drugs can alter Mrp2 expression. Expres-sion may be altered at the level of transcription, translation, and posttranslationthrough endocytic retrieval (9).

3. METHODS (IN VITRO)This chapter outlines several in vitro approaches to study drug transport in

the liver. Although these approaches have been described in detail elsewhere,here we present our perspectives, with particular emphasis on the baculovirusexpression vector system.

3.1. Animal Tissue Preparations

In addition to in vivo studies in animals, several preparations can be used forin vitro experiments. The perfused liver is useful to examine hepatic extrac-tion, metabolism, and biliary excretion, and most closely simulates physiologic

MRPs and Endo- and Xenobiotic Transport 277

Fig. 2. Function of MRP2 in the hepatocyte. Various compounds can be conjugatedby intracellular enzymes including glutathione-S-transferases (GST), UDP-glucuronosyl-transferases (UGT), and sulfotransferases (SULT). Many of these conjugates are theneffluxed across the canalicular membrane in an ATP-dependent manner by MRP2.(Adapted from ref. 10.)

conditions (13). Uptake and/or efflux can be examined in isolated rat or humanhepatocytes. Hepatocyte preparations have been described elsewhere; in addi-tion, they are available from Gentest. Next, it is possible to isolate canalicularand basolateral membrane vesicles, which can then be used for vesicular uptakestudies. The preparation and use of these vesicles has also been described(14,15). Furthermore, the comparison of biliary excretion or canalicularmembrane transport activities between normal rats and Mrp2-deficient rats(Groningen yellow/TR– or Eisai hyperbilirubinemic rats) has facilitated theidentification of Mrp2 substrates and its impact on their transport (16).

3.2. Expression Systems

As shown in Fig. 1, there are several ABC transporters expressed at thecanalicular membrane, and these have overlapping substrating specificities. Oneof the serious limitations of animal tissue preparations is the difficulty in deter-mining the contribution from individual transporters. To overcome this problem,these transporters can be overexpressed to study their functions independently.They can be expressed transiently, by injecting oocytes with cRNA or trans-fecting yeast with an expression plasmid. However, injection of oocytes istedious, and subject to seasonal variations in the harvest of oocytes. Yeastexpression also faces limiting factors, including difficulty harvesting proteinand the low level of posttranslational modifications. Alternatively, transportershave been stably transfected and expressed in several different mammalian celllines, including MDCK cells (17), HeLa cells (18), COS-7 cells (19), CHOcells (20), HEK293 cells (21), and others. Through stable transfections, proteinscan be expressed in a mammalian environment, with appropriate posttransla-tional modifications. However, a long selection process is often required; fur-thermore, MRP2 is not efficiently routed to the plasma membrane (22).

In addition to these methods, virus-mediated expression has been shown tobe an effective approach. Commonly, the genes are inserted into the baculo-virus, which is then used to infect insect cells. Alternatively, genes can beinserted into adenoviruses, and then used to infect certain cell lines. In eithercase, the cell machinery is then conscripted to overexpress the protein of inter-est. Cell membranes can then be harvested, and vesicular uptake experimentscan be performed, as described in Subheading 3.3.1. Several commerciallyavailable products are marketed for baculovirus-mediated expression, whichaccelerate and simplify insertion of the gene of interest into the baculovirusvector. One product with which we have extensive experience is from Gibco(now Invitrogen), called the Bac-to-Bac Baculovirus Expression Vector System,and is described below. In brief, this system involves inserting the cDNA ofinterest into a shuttle vector (pFastBac1), which is then transformed intoDH10Bac cells containing the circular baculovirus genome (bacmid) and a

278 Gerk and Vore

transposition helper plasmid. Transposition takes place, resulting in the inser-tion of the cDNA into the bacmid, and loss of β-galactosidase activity, allow-ing white colonies to be selected on plates containing Blue-gal. Bacmid DNAis then isolated and used to transfect Sf9 cells, which then produce the recom-binant baculovirus. The baculovirus is then amplified, titered, and used to infectSf9 cells for protein production. Another product, from Pharmingen, calledBaculoGold, uses recombination in insect cells under the selective pressure ofantibiotics. More conventional methods have been described in an extensiveand informative manual (23). Most recently, Invitrogen has produced anotherproduct, BaculoDirect, which allows the above mentioned transposition event tooccur enzymatically. This product offers the advantage of speed and conve-nience, but it is quite expensive. More information on these products can befound on the websites for these companies in Table 1.

3.3. Experimental Paradigms

The hypothesis to be tested determines which functional approach is mostappropriate. In this brief overview, we discuss uptake into membrane vesicles,transport across a cell monolayer, and ATPase activity.

3.3.1. Membrane Vesicle Uptake

The preparation and use of membrane vesicles has facilitated numerousinvestigations not possible with living cells, tissues, or organisms. In this invitro preparation, the experimental conditions can, therefore, be modified with-out concerns regarding cell viability. Although preparation of isolated basolat-eral or canalicular membrane vesicles from rat liver is tedious and gives a lowyield, the technique is ideally suited for use with expression systems.

Membrane vesicles are a mixture of inside-out and right-side out orientations,but only the inside-out vesicles have access to ATP and thus can transport thesubstrate, as the ATP-binding cassettes are intracellular-facing. We assumethat ATP diffusion across the membranes is negligible, so the observed ATP-dependent transport represents activity of only the inside-out membrane vesicles.For comparison, membrane vesicles from Sf9 cells are reported to be approx65% inside-out (24); canalicular membrane vesicles are 32 ± 5% inside-out (25);membranes prepared from LLC-PK1 cells are 49% inside-out (26).

Transport experiments are performed in a Tris-sucrose buffer (27), contain-ing ATP or AMP, MgCl2, an ATP-regenerating system, with unlabeled agents ineither dimethyl sulfoxide (0.5%) or vehicle (2% of 10�4�1 Tris-sucrosebuffer–propylene glycol–ethanol). Preliminary studies showed that the choice ofvehicle had no effect on transport. ATP-dependent transport of [3H]E217Ginto membrane vesicles (10 μg/20 μL) is measured by incubating at 37°Cfor 2–5 min in 12 × 75 mm polystyrene tubes (Sarstedt), then stopped with


3.5 mL (a P5000 pipetter is useful for speed) of ice-cold stop buffer (27); themixture is then quickly filtered by transferring to a Millipore 15-mL filterfunnel and collected on 25 mm Durapore 0.4-μm filters (Millipore). Thesefilters were selected due to their minimal binding of E217G at low (90 nM) orhigh (100 μM) concentrations; binding was less than that to HAWP or nitro-cellulose filters, and similar to that to Whatman glass fiber type A/E filters(unpublished data). The tubes are rinsed once with 3.5 mL of stop buffer, thenthe funnel is rinsed again with 3.5 mL of stop buffer, as described (14). Filtersare collected in 7-mL scintillation vials, and radioactivity is dissolved (severalhours or overnight) in scintillation counting cocktail (Bio-Safe II, RPI,Mt. Prospect, IL). Liquid scintillation counting is performed to detect 3H. Dataobtained in the presence of AMP should be subtracted from that in the presenceof ATP; furthermore, data should be corrected for background transport byendogenous transporters. We prefer nonlinear regression for analysis ofMichaelis–Menten saturation curves, and we typically use a weighting schemeof 1/y2, because the variability tends to increase with transport rates.

Similar protocols for MRP1/2 mediated transport into membrane vesiclesare available at www.solvo.com. In addition, this method has recently beenadapted for use in the 96-well format, and is likely to be useful for high-throughput screening for the pharmaceutical industry (28). However, it requiresexpensive equipment and this level of throughput is generally not necessaryfor academic research laboratories.

3.3.2. Transmonolayer Flux

Using cell monolayers derived from tissues of interest (as in the Caco-2,MDCKII or other cell models), one can obtain transcellular transport parameters,which may be helpful in understanding the vectorial transport of substratesacross an epithelium (29,30). Typically, cells are grown to confluence on trans-well filters, and passage of substrate from one side to the other is analyzed,either by liquid scintillation counting, or photometrically. In addition, the use ofa side by side diffusion chamber system (Fig. 3) has been described previouslyin detail (31). The advantage of the latter over the commonly used transwellsystem is the ability to obtain a more accurate estimate of transport parameters(especially for lipophilic compounds) by minimizing the influence of theunstirred layers in both fluid chambers (32,33). The disadvantages includehaving a more complex system of transporters and enzymes to study, and thedifficulty and time required to produce cells grown to confluence on these filters.

3.3.3. ATPase Activity

In addition to transport function, it may also be desirable to examineanother biochemical function of ABC transporters, namely ATPase activity.

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Typically, this is done using membranes prepared from cells expressing theABC transporter of interest. ATPase activity can be used to screen interac-tions between compounds of interest and ABC transporters of concern. How-ever, negative results may not necessarily indicate a lack of interaction, nordoes a positive result indicate that a compound is a transport substrate (34).ATPase activity data can be used to discern the biochemical functions of ABCtransporters (2).

ATPase activity stimulation by a compound is not strictly correlated with itstransport activity (34). For example, a stimulatory (or inhibitory) effect onATPase activity indicates an interaction between the compound and the ABCtransporter; however, a lack of an effect does not indicate a lack of interaction.Part of the discrepancy between ATPase stimulation and transport activity maybe related to the baseline ATPase activity of these transporters. In the absence ofadded substrates, MDR1 exhibits idle ATPase cycling (35), which may occurwith other ABC transporters as well. Idle cycling may make it difficult toobserve a net change in ATPase activity, as follows. If a known transport


Fig. 3. Snapwell diffusion chambers. The chambers consist of the basolateral (left)compartment, the apical (right) compartment, the cells grown on a snapwell filter, andan O-ring to form a seal. Gas is bubbled at 25–30 mL/min through both gas inlet tubes,resulting in a lifting action that creates linear fluid movement through each chamber,thus minimizing the unstirred layers on both sides. Samples can be drawn at any timewithout disruption.

substrate is added to an ABC transporter, it binds to an ABC transporter; ATP ishydrolyzed, and a conformational change occurs resulting in transport; thetransporter may then return to its original state, completing the cycle (2).ATPase activity due to the interaction between a substrate and the ABC trans-porter must be greater than the baseline ATPase activity, to observe a measur-able change in net ATPase activity. However, the presence of added substrate(s)may also alter the rate of ATPase idle cycling, which could result in a decreasedsensitivity of the ATPase assay to detect ATPase stimulation due to transportsubstrates.

ATPase activity is measured by determining the liberation of inorganic phos-phate from ATP with a colorimetric reaction (36). This reaction uses zincacetate and ammonium molybdate to form a complex with inorganic phosphate;the complex is then reduced using ascorbate. We have found this method robustto changes in the incubation buffer, sensitive to low amounts of inorganic phos-phate, and very reproducible. Other detection methods can also be used but arenot discussed here.

The incubation media contains 10 mM MgCl2, 40 mM 2-(N-morpholino)-ethanesulfonic acid, 40 mM tris-(hydroxymethyl)-aminomethane, 5 mM dithio-threitol, 50 mM KCl, 0.1 mM ethylene glycol tetraacetate, 4 mM sodium azide,1 mM ouabain, and 5 mM MgATP, as described (37). The crude protein con-centration is 50 μg/100 μL, and the incubation time is 30 min at 37°C. Theassay is optimized with regards to time and protein by comparing probenecid-stimulated ATPase activity to control (in the case of Mrp2). Reactions arestopped by adding 100 μL of 5% sodium lauryl sulfate, and liberated inorganicphosphate is measured colorimetrically as mentioned above (λ = 850 nm) (36).Data obtained in the presence of vanadate (0.3 mM) are subtracted from eachdetermination to establish the vanadate-sensitive ATPase activity. Appropriatestatistical comparisons can then be performed.

ATPase activity assays have also been adapted for 96-well systems (38). Thelimiting factor with this adaptation is the availability of an accurate and reliableplate reader. This is in part due to the limitations of most plate readers, whichread absorbance at a maximum wavelength of 600 nm, which is below the peaksensitivity of 850 nm (38). Nonetheless, this technique can be used success-fully. Detailed protocols for this method are available at www.gentest.com. orwww.solvo.com.

In conclusion, there are many techniques currently available to study thefunction of hepatic transporters. The key is to choose an appropriate systemfor the experimental questions and hypotheses at hand. Many investigators areusing the baculovirus expression vector system due to its relative ease and lowcost. In the following subheadings are detailed the protocols we have found tobe effective in characterizing Mrp2 function.

282 Gerk and Vore

4. PROTOCOL 1: SF9 CELL CULTURE4.1. Background

Sf9 cells grow well in suspension or attached to plastic. In suspension cul-ture, cells double about every 20–24 h, and viability (by trypan blue exclusion)is >97%. Cells can be seen mostly as singlets and doublets, but a few tripletsand quadruplets (and a few small groups) can be observed. Uncommonly largerclusters can be seen. Cultures having these characteristics (doubling time, via-bility, minimal clumping) are considered “healthy cultures.” Some investiga-tors use “impeller” flasks, in which a magnetic impeller directly contacts theliquid culture. However, these flasks are expensive, difficult to clean, and rela-tively easily contaminated. A more practical and convenient method uses shak-ing cultures, in which cells are grown in polymethylpentene (PMP, availablefrom Nalgene) Erlenmeyer flasks shaken continuously.

4.2. Recovery of Cells From Liquid Nitrogen (or –80°C)

1. Warm 16 mL of the medium to 27°C in a 125-mL PMP flask (Nalgene). We useSF900 II SFM (serum-free medium) from Invitrogen. This medium allows us tomore easily perform transfections and viral plaque assays, both of which must beperformed in the absence of serum. The cost is high, but other common media(such as Grace’s medium with 10% fetal bovine serum) are slightly more expen-sive due to the current high cost of fetal bovine serum. Furthermore, the manu-facturer states that the saturation density in SF900 II SFM is higher than othermedia, potentially allowing a longer log growth phase.

2. Warm the cells to 37°C quickly in a water bath, continually checking to determinewhen the aliquot is nearly melted.

3. Wipe the tube with alcohol, transfer contents to flask.4. Shake at 130–150 rpm on a platform shaker (Lab-Line, available through Fisher)

at 27°C, 1–4 d until cell counts are approx 1.5–2 × 106 cells/mL.5. Split again to 0.6 × 106 cells/mL and allow to grow as described in step 1.6. Split cells again as in step 5, but let them grow to 3–4 × 106 cells/mL.7. Culture as described in Subheading 4.3.

4.3. Routine Subculture

1. The medium is warmed to 27°C.2. Wipe gloved hands and flask with alcohol. Gently swirl flask, and use yellow

1-mL disposable pipets to draw 0.2 mL of cells. Take two samples for each flask,and place in 1.5-mL Eppendorf tubes.

3. Promptly return flask to incubator shaker (27°C, 130–150 rpm).4. Add 0.2 mL of trypan blue to the tube, invert several times to mix.5. Place approx 10 μL of the mix onto the hemacytometer. Under ×10 magnification,

count the total number of cells and the number of dark (dead) cells in the middleset of squares.


6. Count two aliquots per tube, two tubes per flask.7. Determine the total number of cells and the number of dead cells and calculate the

percentage of viable cells. Multiply this percentage by the total number of cells,and then multiply by 5000 to obtain the cell density. Example: 510 total cellscounted, 10 dead cells: 98% viable, 2.5 × 106 viable cells/mL.

8. Seed the cells at 0.6–0.75 × 106 viable cells/mL, 25–50 mL per 125-mL flask, or50–100 mL per 250-mL flask, or 100–200 mL per 500-mL flask.

9. Let the cells grow 2 d, or until they reach a cell density of 3–4.5 × 106 viablecells/mL.

4.4. Cryogenic Storage of Sf9 Cells

1. Centrifuge approx 30 mL of cells (3–4.5 × 106 viable cells/mL) at 100 RCF for5 min.

2. Aspirate supernatant, resuspend to 1–1.5 × 107 viable cells/mL in 7.5% DMSO–92.5% SFM II 900 medium chilled on ice.

3. Aliquot into 1-mL cryovials labeled with the date, passage number, and type ofcells. Place at –80°C overnight in cryogenic freezing container (Nalgene), thentransfer to liquid nitrogen if desired, or retain at –80°C. Our experience shows thatcells retain viability even after several months at –80°C.

5. PROTOCOL 2: SUCROSE-FRACTIONATED SF9 MEMBRANEVESICLE PREPARATION FROM CELLS INFECTED WITH MRP2-BACULOVIRUS

Steps 1–3 are based on ref. 39 and steps 4–20 are adapted from ref. 27.

1. Grow Sf9 cells in suspension culture with Gibco SFM II-900 serum-free insectcell medium to a concentration of 3–4 × 106 viable cells/mL.

2. Infect cells with titered viral stock (107–108 pfu/mL) at multiplicity of infection = 3,as 80- to 100-mL cultures containing 1 × 106 viable cells/mL, in 250-mL poly-methylpentene (PMP) screw-capped flasks.

3. At 64–68 h postinfection, harvest 1.5–2 × 108 Sf9 cells by centrifugation (500g,4°C, 5 min) using 200-mL Nalgene conical centrifuge tubes.

4. Withdraw or aspirate supernatant, and resuspend pellet in 10 mL of hypotoniclysis buffer (1 mM TrisCl, 0.1 mM EDTA, pH 7.4, at 4°C, with 1 mM phenyl-methylsulfonyl fluoride (stock = 100 mM in isopropyl alcohol), 5 μg/mL of leu-peptin, 1 μg/mL of pepstatin (stock = 5 mg/mL in dimethyl sulfoxide), 5 μg/mLof aprotinin.

5. Determine the total volume of cell suspension minus 10 mL to estimate thevolume of the cell pellet. Add more lysis buffer to reach a total volume of 40times the cell pellet volume.

6. Incubate cells on ice for 60 min with gentle occasional swirling. Subsequent stepsare carried out in the cold room at 4°C.

7. Centrifuge cell lysate at 27,500 rpm in a Beckman SW28 rotor (100,000gav) at4°C for 40 min.

284 Gerk and Vore

8. Withdraw or aspirate supernatant, resuspend the pellet in 10 mL of isotonic TSbuffer containing 10 mM Tris-Cl, 250 mM sucrose, pH 7.4, at 4°C.

9. Homogenize 10–15 mL in a 15-mL Dounce B tight homogenizer (glass/glass,tight pestle, 30 strokes = cycles) on ice in the cold room. Be careful not to intro-duce air bubbles.

10. Slowly, gently, and carefully overlay homogenate on 2 mL of 38% (w/v) sucrosesolution in 5 mM Tris-HEPES, pH 7.4, at 4°C in 12 mL of Beckman Ultra-Clearcentrifuge tubes. Bring the volume to the bottom of the word “Beckman” on thetubes with homogenate or TS buffer.

11. Carefully place tubes in SW41 or TH641 rotor and centrifuge for 60 min at39,000 rpm (255,000g) at 4°C.

12. Collect the turbid layer at the interface and dilute to 35 mL with TS buffer andcentrifuge 27,500 rpm (100,000g) for 40 min at 4°C in an SW28 rotor. Alterna-tively, dilute to 23–24 mL and load into tubes for the SW41 rotor and centrifugefor 40 min at 4°C at 100,000g.

13. Aspirate supernatant, resuspend with 300–400 μL of TS buffer first using P1000pipet, then connect P200 pipet tip to the end of P1000 tip until all the mass is uni-formly resuspended.

14. Vesiculate by passing the suspension 30 times (= 15 cycles) through a 25-gaugeneedle on the end of a 1-mL syringe. Avoid making bubbles or foam.

15. Aliquot into labeled tubes and drop tubes into liquid nitrogen for about 5–10 min,then transfer to –80°C.

16. Measure protein concentration by the Lowry method, bovine albumin as the stan-dard. Usually protein concentration is about 3–5 μg/μL or higher.

17. Total processing time from infected cells in suspension culture to vesicles in liquidnitrogen should be about 6 h or less.

18. To use membranes, remove from –80°C and place in 37°C water bath for 1–2 min,then put on ice for at least 2 min.

19. If desired, revesiculate by passing vesicles diluted to 2.5 μg/μL again 30 timesthrough a 25-gauge needle as described in step 14. This part is not mentionedin the literature for Sf9 vesicles but it is commonly performed for livermembrane vesicles. We include this every time and obtain reproducible results(CV ± 10%).

20. Keep the tube on ice and complete the experiments within 2–3 h after thawing.Use 4 μL to deliver 10 μg per replicate. Before withdrawing each sample, gentlyflick the tube two or three times to stir the membranes, to improve uniformity.

21. We typically use the membranes within 3 wk of production. However, SolvoBiotechnology (Budapest, Hungary) adds protease inhibitors and extends storageat –80°C to 2 yr.

6. PROTOCOL 3: SF9 CRUDE MEMBRANE VESICLEPREPARATION FROM BACULOVIRUS-INFECTED CELLS

1. Grow Sf9 cells in suspension culture with Gibco SFM II-900 serum-free insectcell medium to a concentration of 3–4 × 106 viable cells/mL.


2. Infect cells with titered viral stock (≥108 pfu/mL) at an appropriate multiplicity ofinfection (MOI), as 80- to 100-mL cultures containing 1 × 106 viable cells/mL, in250-mL polymethylpentene (PMP) screw-capped flasks (Nalgene).

3. At the desired time post-infection, harvest 1.5–2 × 108 Sf9 cells by centrifugation(500g, 4°C, 5–6 min) using 200-mL Nalgene conical centrifuge tubes.

4. Withdraw or aspirate supernatant, and resuspend pellet in 10 mL of hypotoniclysis buffer (1 mM Tris-Cl, 0.1 mM ethylenediamine tetraacetate, pH 7.4, at 4°C,with 1 mM phenylmethylsulfonyl fluoride (stock = 100 mM in isopropyl alco-hol), 5 μg/mL of leupeptin, 1 μg/mL of pepstatin (stock = 5 mg/mL in dimethylsulfoxide), and 5 μg/mL of aprotinin.

5. Determine the difference in the total volume of cell suspension minus 10 mL toestimate the volume of the cell pellet. Multiply the total volume of cell pellets by40 and add more lysis buffer to reach this value in milliliters.

6. Incubate cells on ice for 60 min with gentle occasional swirling.7. Homogenize 10–15 mL in a 15-mL Dounce B tight homogenizer (glass/glass,

tight pestle, 30 strokes = cycles) on ice. Be careful not to introduce air bubbles.8. Centrifuge cell lysate at 500g in a 15- or 50-mL conical bottom tube at 4°C for

5–6 min.9. Transfer supernatant to ultracentrifuge tubes and centrifuge at 100,000gav

(25,000 rpm in an SW41 rotor, or 27,500 rpm in an SW28 rotor) 30 min at 4°C.10. Aspirate supernatant, resuspend the pellet with 300–400 μL of TS buffer first

using a P1000 pipet, then connect P200 pipet tip to the end of P1000 tip until allthe mass is uniformly resuspended.

11. Vesiculate by passing the suspension 30 times (= 15 cycles) through a 25-gaugeneedle on the end of a 1-mL syringe.

12. Aliquot into labeled tubes and drop tubes into liquid nitrogen for about 5–10 min,then transfer to –80°C.

13. Measure protein concentration by the Lowry method (40), using bovine albuminas the standard. Usually protein concentration is about 6–10 μg/μL or higher.

14. Total processing time from infected cells in suspension culture to vesicles in liquidnitrogen should be about 5 h or less.

15. To use membranes, remove from –80°C and place in 37°C water bath for 1–2 min,then put on ice for at least 2 min.

16. If desired, revesiculate by passing diluted vesicles again 30 times through a25-gauge needle as above.

17. Keep the tube on ice and use within 2–3 h after thawing. Before withdrawingeach sample, gently flick the tube two or three times to stir the membranes, tohelp with uniformity.

7. SUMMARY AND CONCLUSIONSTransport of endo- and xenobiotics is an important hepatocellular function,

which serves two roles: bile formation and excretion. The MRPs in particularmediate the efflux of diverse sulfate, glutathione, or glucuronide conjugates ofendogenous compounds like estrogen and bilirubin, as well as drugs and toxins

286 Gerk and Vore

from the hepatocyte. MRP2 in particular contributes importantly to both excre-tion and bile formation. Several in vitro approaches may be used to character-ize hepatic transport processes. Because the main hepatic transporters havebeen cloned, it is possible to examine the function and mechanism of isolatedtransporters in expression systems. This chapter particularly emphasizes use ofthe baculovirus expression vector system to express MRP2 in Sf9 insect cells.

REFERENCES1. Stein WD. Channels, Carriers, and Pumps: An Introduction to Membrane Trans-

port. San Diego: Academic Press; 1990.2. Holland IB, Blight MA. ABC-ATPases, adaptable energy generators fuelling trans-

membrane movement of a variety of molecules in organisms from bacteria tohumans. J Mol Biol 1999;293:381–399.

3. Stein WD. Transport and Diffusion Across Cell Membranes. San Diego: AcademicPress; 1986.

4. Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC) trans-porter superfamily. Genome Res 2001;11:1156–1166.

5. Borst P, Elferink RO. Mammalian ABC transporters in health and disease. AnnuRev Biochem 2002;71:537–592.

6. Faber KN, Muller M, Jansen PL. Drug transport proteins in the liver. Adv DrugDeliv Rev 2003;55:107–124.

7. Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function,and regulation. Physiol Rev 2003;83:633–671.

8. Ballatori N, Rebbeor JF. Roles of MRP2 and oatp1 in hepatocellular export ofreduced glutathione. Semin Liver Dis 1998;18:377–387.

9. Gerk PM, Vore M. Regulation of expression of the multidrug resistance-associated protein 2 (MRP2) and its role in drug disposition. J Pharmacol ExpTher 2002;302:407–415.

10. Keppler D, Leier I, Jedlitschky G. Transport of glutathione conjugates and glucu-ronides by the multidrug resistance proteins MRP1 and MRP2. Biol Chem 1997;378:787–791.

11. Shoda J, Kano M, Oda K, et al. The expression levels of plasma membrane trans-porters in the cholestatic liver of patients undergoing biliary drainage and theirassociation with the impairment of biliary secretory function. Am J Gastroenterol2001;96:3368–3378.

12. Kullak-Ublick GA, Stieger B, Hagenbuch B, Meier PJ. Hepatic transport of bilesalts. Semin Liver Dis 2000;20:273–292.

13. Huang L, Smit JW, Meijer DK, Vore M. Mrp2 is essential for estradiol-17β(β-D-glucuronide)-induced cholestasis in rats. Hepatology 2000;32:66–72.

14. Boyer JL, Meier PJ. Characterizing mechanisms of hepatic bile acid transportutilizing isolated membrane vesicles. Methods Enzymol 1990;192:517–533.

15. Meier PJ, Boyer JL. Preparation of basolateral (sinusoidal) and canalicular plasmamembrane vesicles for the study of hepatic transport processes. Methods Enzymol1990;192:534–545.


16. Suzuki H, Sugiyama Y. Transporters for bile acids and organic anions. PharmBiotechnol 1999;12:387–439.

17. Evers R, de Haas M, Sparidans R, et al. Vinblastine and sulfinpyrazone export bythe multidrug resistance protein MRP2 is associated with glutathione export. Br JCancer 2000;83:375–383.

18. Loe DW, Almquist KC, Cole SP, Deeley RG. ATP-dependent 17 β-estradiol 17-(β-D-glucuronide) transport by multidrug resistance protein (MRP). Inhibition bycholestatic steroids. J Biol Chem 1996;271:9683–9689.

19. Ryu S, Kawabe T, Nada S, Yamaguchi A. Identification of basic residues involvedin drug export function of human multidrug resistance-associated protein 2. J BiolChem 2000;275:39617–39624.

20. Shapiro AB, Ling V. ATPase activity of purified and reconstituted P-glycoproteinfrom Chinese hamster ovary cells. J Biol Chem 1994;269:3745–3754.

21. Hagmann W, Nies AT, Konig J, Frey M, Zentgraf H, Keppler D. Purification of thehuman apical conjugate export pump MRP2 reconstitution and functional charac-terization as substrate-stimulated ATPase. Eur J Biochem 1999;265:281–289.

22. Borst P, Evers R, Kool M, Wijnholds J. The multidrug resistance protein family.Biochim Biophys Acta 1999;1461:347–357.

23. O’Reilly DR, Miller LK, Luckow VA. Baculovirus expression vectors: a laboratorymanual. New York: Oxford University Press; 1994.

24. van Aubel RA, van Kuijck MA, Koenderink JB, Deen PM, van Os CH, Russel FG.Adenosine triphosphate-dependent transport of anionic conjugates by the rabbitmultidrug resistance-associated protein Mrp2 expressed in insect cells. MolPharmacol 1998;53:1062–1067.

25. Bohme M, Muller M, Leier I, Jedlitschky G, Keppler D. Cholestasis caused byinhibition of the adenosine triphosphate-dependent bile salt transport in rat liver.Gastroenterology 1994;107:255–265.

26. Konno T, Ebihara T, Hisaeda K, et al. Identification of domains participating inthe substrate specificity and subcellular localization of the multidrug resistanceproteins MRP1 and MRP2. J Biol Chem 2003;278:22908–22917.

27. Ito K, Suzuki H, Sugiyama Y. Single amino acid substitution of rat MRP2 results inacquired transport activity for taurocholate. Am J Physiol 2001;281:G1034–G1043.

28. Tabas LB, Dantzig AH. A high-throughput assay for measurement of multidrugresistance protein-mediated transport of leukotriene C4 into membrane vesicles.Anal Biochem 2002;310:61–66.

29. Akita H, Suzuki H, Ito K, et al. Characterization of bile acid transport mediatedby multidrug resistance associated protein 2 and bile salt export pump. BiochimBiophys Acta 2001;1511:7–16.

30. Cui Y, Konig J, Keppler D. Vectorial transport by double-transfected cells express-ing the human uptake transporter SLC21A8 and the apical export pump ABCC2.Mol Pharmacol 2001;60:934–943.

31. Hidalgo IJ, Hillgren KM, Grass GM, Borchardt RT. A new side-by-side diffusioncell for studying transport across epithelial cell monolayers. In Vitro Cell Dev Biol1992;28A:578–580.

288 Gerk and Vore

32. Hidalgo IJ, Hillgren KM, Grass GM, Borchardt RT. Characterization of theunstirred water layer in Caco-2 cell monolayers using a novel diffusion apparatus.Pharm Res 1991;8:222–227.

33. Yu H, Sinko PJ. Influence of the microporous substratum and hydrodynamics onresistances to drug transport in cell culture systems: calculation of intrinsic trans-port parameters. J Pharm Sci 1997;86:1448–1457.

34. Polli JW, Wring SA, Humphreys JE, et al. Rational use of in vitro P-glycoproteinassays in drug discovery. J Pharmacol Exp Ther 2001;299:620–628.

35. Litman T, Druley TE, Stein WD, Bates SE. From MDR to MXR: new under-standing of multidrug resistance systems, their properties and clinical significance.Cell Mol Life Sci 2001;58:931–959.

36. Saheki S, Takeda A, Shimazu T. Assay of inorganic phosphate in the mild pHrange, suitable for measurement of glycogen phosphorylase activity. Anal Biochem1985;148:277–281.

37. Bakos E, Evers R, Sinko E, Varadi A, Borst P, Sarkadi B. Interactions of the humanmultidrug resistance proteins MRP1 and MRP2 with organic anions. Mol Pharmacol2000;57:760–768.

38. Drueckes P, Schinzel R, Palm D. Photometric microtiter assay of inorganic phos-phate in the presence of acid-labile organic phosphates. Anal Biochem 1995;230:173–177.

39. Van Aubel RA, Peters JG, Masereeuw R, Van Os CH, Russel FG. Multidrug resis-tance protein mrp2 mediates ATP-dependent transport of classic renal organicanion p-aminohippurate. Am J Physiol 2000;279:F713–F717.

40. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with thefolin phenol reagent. J Biol Chem 1951;193:265–275.


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