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Multidrug Resistance Protein 2Transport Properties o f a Drug Efflux Pump

R.A.M.H. van Aubel

Multidrug Resistance Protein 2 Transport Properties o f a Drug Efflux Pump R.A.M.H. van Aubel. Thesis University of Nijmegen -With Ref.- With summary in dutch

ISBN 90-9013589-8

© 2000 by R.A.M.H. van Aubel

The studies presented in this thesis were performed at the Departments of Cell Physiology and Pharmacology & Toxicology, Faculty of Medical Sciences, University of Nijmegen, The Netherlands


Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen

Proefschrift

ter verkrijging van de graad van doctor aan de Katholieke Universiteit Nijmegen, volgens besluit van het College van Decanen in het openbaar te verdedigen op dinsdag 9 Mei 2000 des namiddags om 1.30 uur precies

door

Raimundus Albertus Martinus Hermanus van Aubel

geboren op 23 juli 1970 te Schaesberg

Promotoren:Prof. Dr. F.G.M. Russel Prof. Dr. C.H. van Os

Manuscriptcommissie:Prof. Dr. J.J.H.H.M. de Pont Prof. Dr. E.J.J. van Zoelen Dr. M. Müller (RUG)


Contents

Abbreviations

Chapter 1Introduction: molecular pharmacology of renal organic anion transporters

Chapter 2Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance protein Mrp2 expressed in insect cells

Chapter 3Immunolocalization of the multidrug resistance protein Mrp2 in rabbit small intestine, liver, and kidney

Chapter 4Mechanisms and interaction of vinblastine and reduced glutathione transport in membrane vesicles by the rabbit multidrug resistance protein Mrp2 expressed in insect cells

Chapter 5The multidrug resistance protein Mrp2 mediates ATP-dependent transport of the classical organic anion p-aminohippurate

Chapter 6Summary, general conclusions and future perspectives

Samenvatting en conclusies

List of publications

Dankwoord

9

11

49

69

87

105

119

129

137

139

Curriculum Vitae 141

Abbreviations

ABC ATP-binding cassetteANOVA analysis of variance5'-AMP adenosine 5'-monophosphateATP adenosine triphosphateBBM brush-border membraneBLM basolateral membraneBSA bovine serum albuminCDDP c/s-diamminedichloroplatincDNA complementary DNACFTR cystic fibrosis transmembrane conductance regulatorcMOAT canalicular multispecific organic anion transporterCsA cyclosporin ADMSO dimethylsulphoxideDNP-SG 5-(dinitrophenyl)-glucuronideDTT dithiothreitolE2175G 17fi-estradiol-17-5-D-glucuronideEDTA ethylenediamine tetraacetateEHBR Eisai hyperbilirubinemic rat (Mrp2-deficient Sprague Dawley rat)FL fluoresceinFL-MTX fluorescein-methotrexateFURA-AM fura-2 acetoxy methyl esterGSH reduced glutathioneGSSG oxidized glutathioneGY/TR- Groningen yellow/ Transport deficient rat (Mrp2-deficient Wistar rat)HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acidIgG immunoglobulin Gkb kilobaseskDa kilodaltonsKi inhibition constantKm Michaelis-Menten constantLTC4 leukotriene C4LY lucifer yellowmAb monoclonal antibodyMDCKII type II (proximal tubule derived) kidney cell line of Madin-Darby canineMDR multidrug resistance

MK571 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethyl-amino-3- oxopropyl)-thio}-methyl]thio)propanoic acid

MOI multiplicity of infectionMRP multidrug resistance proteinMTX methotrexateOA organic anionPAGE polyacrylamide gel electrophoresisPAH para-aminohippuratePBS phosphate-buffered salinePgp P-glycoproteinPLP periodate lysine paraformaldehydePMSF phenyl methyl sulphonyl fluoridePNGase F N-glycosidase FPT Proximal tubuleSD standard deviationSDS sodium dodecyl sulphateSE standard error of the meanSf9 Spodoptera frugiperda cellsTBS tris-buffered salineTS tris sucroseTris Tris(hydroxymethyl)aminomethaneTween 20 polyoxyethylene sorbitan monolaurateVBL vinblastineVCR vincristineVmax maximum rate of transport

Chapter 1

Introduction: Molecular Pharmacology of Renal Organic

Anion Transporters

Remon A.M.H. van Aubel, Rosalinde Masereeuw, Frans G.M. RusselDepts. of Pharmacology and Toxicology, University of Nijmegen, The Netherlands

American Journal of Physiology (Renal Physiology), 2000, in press (adapted version)

Summary...................................................................................................................... 13

Introduction.................................................................................................................. 14

Basolateral transport systems......................................................................................... 21Organic anion transporter Oat1 (PAH/dicarboxylate exchanger).......................... 22Oat2 and Oat3/OAT3......................................................................................... 23Sulfate/anion exchanger Sat1 ...............................................................................24Multidrug resistance protein MRP1......................................................................24MRP3, MRP4, MRP5 and MRP6...........................................................................25

Intracellular disposition of anionic drugs........................................................................26

Apical transport systems................................................................................................ 28MRP2 ................................................................................................................ 29Organic anion transport polypeptides Oatp1 and Oatp3...................................... 31Organic anion transporters Oat-k1 and Oat-k2..................................................... 31Na+/nucleoside cotransporters CNT1 and CNT2..................................................32H+/peptide cotransporters PEPT1 and PEPT2.......................................................33

Conclusive remarks....................................................................................................... 33Aim and Outline of this Thesis...................................................................................... 34

References.................................................................................................................... 35

11

Molecular Pharmacology of Renal Organic Anion Transporters

Summary

Renal organic anion transport systems play an important role in the elimination of drugs, toxic compounds and their metabolites, many of which are potentially harmful to the body. The renal proximal tubule is the primary site of carrier mediated transport from blood to urine of a wide variety of anionic substrates. Recent studies have shown that organic anion secretion in renal proximal tubule is mediated by distinct sodium-dependent and sodium-independent transport systems. Knowledge of the molecular identity of these transporters and their substrate specificity has increased considerably in the past few years by cloning of various carrier proteins. However, a number of fundamental questions still have to be answered to elucidate the participation of the cloned transporters in the overall tubular secretion of anionic xenobiotics. This review summarizes the latest knowledge on molecular and pharmacological properties of renal organic anion transporters and homologs, with special reference to their nephron and plasma membrane localization, transport characteristics, and substrate and inhibitor specificity. A number of the recently cloned transporters, such as the PAH/dicarboxylate exchanger OAT1, the anion/sulfate exchanger SAT1, the peptide transporters PEPT1 and PEPT2, and the nucleoside transporters CNT1 and CNT2, are key proteins in organic anion handling that possess the same characteristics as has been predicted from previous physiological studies. The role of other cloned transporters, such as MRP1, MRP2, OATP1, OAT-K1, and OAT-K2, is still poorly characterized, whereas the only information that is available on the homologs OAT2, OAT3, OATP3, and MRP3-6, is that they are expressed in the kidney, but their localization, and not to mention their function remain to be elucidated.

(available online at http://ajprenal.physiology.org/)

13

http://ajprenal.physiology.org/

Chapter One (Introduction)

Introduction

The human body is continuously exposed to a great variety of xenobiotics, via food, drugs, occupation and environment. Excretory organs such as kidney, liver and intestine defend the body against the potentially harmful effects of these compounds by biotransformation into less active metabolites and excretory transport processes. Most drugs and environmental toxicants are eventually excreted into the urine, either in the unchanged form or as biotransformation products. The mechanisms that contribute to their renal excretion are closely related to the physiological events occurring in the nephrons, i.e. filtration, secretion and reabsorption. Carrier-mediated transport of xenobiotics and their metabolites is confined to the proximal tubule, and separate carrier systems exist for the active secretion of organic anions and cations. Both systems are characterized by a high clearance capacity and tremendous diversity of substances accepted, probably resulting from multiple transporters with overlapping substrate specificities.

This review will focus on the molecular aspects of renal organic anion transporters. These systems play a critical role in the elimination of a large number of drugs (e.g. antibiotics, chemotherapeutics, diuretics, nonsteroidal antiinflammatory drugs, radiocontrast agents, cytostatics), drug metabolites (especially conjugation products with glutathione, glucuronide, glycine, sulfate, acetate), and toxicants and their metabolites (e.g. mycotoxins, herbicides, plasticizers, glutathione S-conjugates of polyhaloalkanes, polyhaloalkenes, hydroquinones, aminophenols), many of which are specifically harmful to the kidney. For a review on the molecular pharmacology of renal organic cation transporters the reader is referred to a recent comprehensive paper by Koepsell et al. [80]. The purpose of this paper is to give a concise review of the latest molecular information on organic anion transporters that contribute to the renal handling of xenobiotics and their metabolites. These transporters are depicted in Figure 1 and listed in Tables 1 and 2. The systems involved in organic anion secretion can be functionally

14


subdivided in the well characterized, sodium-dependent p-aminohippurate (PAH) system and a recently discovered sodium-independent system [108,109,147]. Both systems mediate two membrane translocation steps arranged in series: uptake from blood across the basolateral membrane of renal epithelial cells followed by efflux into urine across the apical membrane. While transported through the cytoplasm, substrates for both systems also accumulate in intracellular compartments. Furthermore, at the apical membrane several transport systems have been identified which are involved in reabsorption of anionic xenobiotics.

Recent discoveries on the molecular identity of some of the renal organic anion transporters (OAT1, SDCT2, SAT1, PEPT1, PEPT2, CNT1, CNT2) have confirmed the characteristics that have been predicted creatively by earlier functional studies performed in isolated membrane vesicles and proximal tubules. The same studies have indicated the existence of further anion transporters that have not yet been identified at the molecular level. For other recently cloned organic anion transporters information on their function (MRP1, MRP2, OATP1, OAT-K1, OAT-K2) and renal localization (OAT2, OAT3, MRP3-6) remains to be elucidated. Emphasis in this review is being placed on molecular characteristics, nephron and plasma membrane localization, transport properties, and substrate and inhibitor specificity of cloned renal organic anion transporters and homologs.

15

Table 1Molecular characteristics o f organic anion transporters in the kidneya

Name Alternativename

Species Acc. No. Chromosomelocalization

Mrb(kDa)

mRNA(kb)

TM TissueDistribution

NephronDistribution

MembraneLocalization

References

OAT1 hPAHT/ human AF097490/AB009697/ 11q11.7 60 2.4 12 k PT BLM [59,105,150,152]hROATI AF057039/AF104038

Oat1 ROAT1 rat AB004559/AF008221 60 2.4 12 k PT (S2) BLM [166,176,186,191]Oat1 fROAT flounder Z97028 60 nd 12 nd nd nd [205]Oat1 NKT/mOAT mouse U52842 19 60 2.5 11 k PT nd [89,103]

Oat2 NLT rat L27651 60 2.1 12 k, li nd nd [165,172]

OAT3 human AF097491 11q11.7 62 3.0 12 k nd nd [150]Oat3 Roct mouse AF078869 19 62 2.1 12 k PT nd [13]Oat3 rat AB017446 k, li, b, e nd nd [88]

Sat1 rat L23413 75 3.7 12 k, li, b, m PT (S1-S3) BLM [9,73]

MRP1 M RP human L05628 16p13.12-13 171 6.5 18 ubiquitous nd nd [20,39,213]Mrp1 mouse AF022908 171 6.5 18 ubiquitous LH, CCD BLM [174,201,203]

MRP2 cMOAT/ human NM_000392/ 10q24 175 6.5 18 k, li, si, n nd nd [82,181]cM RP U63970/ U49248/

X96395Mrp2 rat L49379/ D86086/ 175 6.5 18 k, li, si PT (S1-S3) BBM [75]

X96393Mrp2 rabbit Z49144 175 6.5 18 k, li, si PT (S1-S3) BBM [194], unpublished

MRP3 MOAT-D/ human Y17151/ AF104943 17q21.3 170 6.5 18 k(?), li, si, c, lu, nd BLM c [42,78,82,84,188]cMOAT2 NM_003786/ p

AF085690/ AF009670Mrp3 MLP2 rat AB010887/ AF072816 170 6.5 18 k, si, c, lu nd nd [57]

MRP4 MOAT-B human U83660/ AF071202 13q31-32 150 6.0 12 (k, lu, bl, gb)d nd nd [82]ubiquitous6 [90]

MRP5 MOAT-C/ human U83661/ AF104942/ 3q27 160 nd 12 ubiquitous nd BLM [5,82,118,202]SMRP/ AB005659/AB019002pABC11 /AF146074

Mrp5 mouse AB019003 160 nd 12 nd nd nd unpublished

MRP6 MOAT-E human AF076622 16p13 165 6.0 18 k, li nd nd [6,83]Mrp6 MLP1 rat AB010466/ U73038 165 6.0 18 k, li, du nd BLM c [57,107]Mrp6 mouse AB028737 165 nd 18 nd nd nd unpublished

Name Alternativename

Species Acc. No. Chromosomelocalization

Mrb(kDa)

mRNA(kb)

TM TissueDistribution

NephronDistribution

MembraneLocalization

References

Oatpl Oatp rat L19031 nd 74 2.7 12 k, li, b, lu, m, c PT (S3) BBM [8,68]

Oatp3 rat AF041105 nd 74 2.8/3.9 12 k, r nd nd [1]

Oat-k1 rat D79981 nd 74 3.0 12 k PT (S3) BBM [117,158]

Oat-k2 rat AB012662 nd 55 2.5 8 k PT (S1-S3)/ CCD BBM [114]

CNT1 human U62968 15q25-26 71 3.4 14 k, si, li nd nd [153]Cnt1 cNT1 rat U10279 71 3.0 14 k, si, li nd nd [38,60]Cnt1 pig AF009673 71 nd 14 nd nd nd [137]

CNT2 hSPNT1 human U84392 15q15 71 4.5 14 ubiquitous nd nd [154,198]Cnt2 SPNT rat U25055 71 3.5 14 k, si, li nd nd [18,38]Cnt2 mouse AF079853 71 nd 14 nd nd nd unpublished

PEPT1 human AF043233/ U13173/ 13q33-34 78 3.3 12 k, si, li, pl nd nd [95]U21936

PepT1 rat D50306/ D50664 78 3.0 12 k, si PT (S1-S2) BBM [126,159,169]PepT1 mouse Y07561 78 nd 12 nd nd nd unpublishedPepT1 rabbit U06467/ U13707 78 2.9 12 k, si, li, b cortex nd [11,37]

PEPT2 human S78203 3q13.3-q21 78 4.2 12 k, b nd nd [151]PepT2 rat D63149 78 4.0 12 k, b, lu PT (S3) BBM [160,169,197]PepT2 rabbit U32507 78 4.8 12 k, b, lu, li nd nd [10]

“Abbreviations: (R)OAT: (renal) organic anion transporter; hPAHT: human PAH transporter; NKT: novel kidney transporter; NLT: novel liver transporter; Sat1 : sulfate anion transporter; MRP: multidrug resistance protein; cMOAT: canalicular multispecific organic anion transporter; cMRP: canalicular M RP; MLP: multidrug resistance protein-like protein; Oatp: organic anion transport polypeptide; Oat-k1/Oat-k2: kidney-specific organic anion transporter 1 and 2; CNT: concentrative nucleotide transprorter: PEPT: peptide transporter; TM: transmembrane-spanning domain; BLM: basolateral membrane; BBM : brush-border membrane; PT: proximal tubule; nd: not determined; k: kidney; li: liver; lu: lung; b: brain; si: small intestine; c: colon; t: testis; r: retina; pl: placenta; p: pancreas; n: nerve; du: duodenum; m: muscle; bl: bladder; gb: gallbladder; CCD: cortical collecting duct; LH: limb of Henle's loop

bCalculated from the deduced amino acid sequence. cBased on localization in liver (baso)lateral membranes. dTissue distribution reported by Kool et al. [82].'Tissue distribution reported by Lee et al. [90].

Table 2Functional characteristics o f organic anion transporters in the kidneya bName transport

mechanismSubstrates (Km)

unconjugated_______________ conjugated

Inhibitors (Ki) References

OAT 1

Oat1

human

rat

OA/dicarboxylateantiport

OA/dicarboxylateantiport

Oat2

Oat3

Sat1

rat

rat

nd

nd

rat OA/ sulfateantiport

Mrp2 rat

PAH (5 uM)

PAH (14-70 uM); salicylate; MTX; cAMP; acetylsalicylate; indomethacin; folate; cGMP; PGE2; urate; a- KG; ochratoxin A

furosemide; indomethacin; probenecid; urate; a-KG; glutarate

naproxen (2 uM); ibuprofen (3.5 uM); salicylurate (11 uM); piroxicam (52 uM); salicylate (341 uM); acetylsalicylate (428 uM); phenacetin (488 uM); paracetamol (2 mM); furosemide; indomethacin (10 uM); probenecid; urate; a-KG; glutarate; MTX; PGE2; cAMP; cGMP

[59,105,150]

[3,166,176,187,191]

MRP1 human primary active

(18 uM); salicylate (90 bumetanide; enalapril;uM); acetylsalicylate cefoperazone; cholate

PAH (65 uM); BSP; probenecid; indocyanineochratoxin A (0.74 uM); green; bumetanide; piroxicam;estrone-sulfate (2.3 uM); furosemide; AZT; DlDScimetidine

sulfate (320 uM); DIDS; oxalate; sulfateoxalate

GSH; PAH LTC4 (0.1 uM); E2 1 7 KG (1.5 MK571 (0.6 uM); CsA (5 uM);uM); DNP-SG (3.6 uM); PSC833 (27 uM); S-(decyl)-bilirubin-glucuronide; GSSG glutathione (0.7uM); GS-DOX(93 uM); AFB1-SG (0.2 uM); (60/120 nM); GS-DAUPGA 1-SG; PGA2-SG (1 uM); (70/200 nM); probenecidetoposide-glucuronide; S-(ethacryn ic acid)-glutathione

[165]

[88]

[9,73]

[33,35,69,70, 76,92, 98,99, 101,145,149, 210]

MRP2 human primary active GSH; MTX LTC4(1 uM); E217KG (7 uM); [24,34]DNP-SG; PGA 1-SG; bilirubin-glucuronide; S- (ethacryn ic acid)-glutathione

GSH; MTX (100 uM); LTC4(1 uM); E217SG (7 uM); MK571; CsA; GSH (20 mM) [2,24,63,65,folate; pravastatin (170 LTD4(1.5 uM); N-acetyl- LTE4 67,70, 79, 87,uM); BSP (31 uM); Fluo- (5.2 uM); DNP-SG (0.1 8 uM); 106,113,130,3 (4 uM); BQ123 (100 bilirubin-glucuronide; GSSG 134-136,142,uM); BQ485 (7 uM); BQ518 (15 uM);

(111 uM); p-nitrophenyl- glucuronide (20 uM); a-

194, 206]

temocaprilat (100 uM); naphthyl-^-D-glucuronide;cefpiramide; ceftriaxone E3040-glucuronide

Mrp2 rabbit GSH LTC4(1 uM); E217SG PAH; lucifer yellow;fluorescein-methotrexate;phenolphthalein-glucuronide;CsA, probenecid

MRP3 human primary active Mrp3 rat



Mrp6 rat primary active

MTXMTX

PMEA; AZTMP

CMFDA; BCECF; FDA

BQ123

DNP-SGE217KG (70 uM); E3040- glucuronide

E3040-glucuronide;a-naphthyl-j3-D-glucuronide;MTX

dipyridamole

sulfinpyrazone

[192-194]

[84][58]

[164]

[118]

[1 0 7 ]

18

Name transport Substrates (Km) Inhibitors (Ki) Referencesmechanism

unconjugated conjugatedOatpl rat OA/GSH TC (27 uM); BSP (1.5 uM); LTC4 (0.27 j j M ); DNP-SG [32,41,64,71,

antiport aldosterone (15 nM); (408 ̂ M); E217ßG (3 ^M); 72,94,138]cortisol (13 nM); ouabain estrone-sulfate (4.5 >wM)(17 mM); ochratoxin A(17 uM); thyroxine;triiodo-L-thyronine;enalapril (214 uM);temocrapilat (47 uM)

Oatp3 rat nd TC (18 uM); thyroxine (5 [1]uM); triiodo-L-thyronine (7uM)

Oat-k1 rat nd MTX (1 uM); folate MTX; BSP; probenecid; PAH; [116,158]furosemide; valproate; DIDS;TCA; ibuprofen; flufenamate;phenylbutzone; indomethacin(1 mM); ketoprofen (2 mM)

Oat-k2 rat nd TC (10 ywM); MTX; PGE2; MTX; BSP; probenecid; PAH; [114]folate furosemide; valproate; DIDS;

TCA; levofloxacin;indomethacin; testosteron;dexamethasone; 17^-estradiol;ouabain

CNT1 human nucleoside/ adenosine (15/26 uM); AZT; cytarabine; [36,60,153,Cnt1 rat Na+ symport thymidine (13 uM); dideoxycytidine; floxidine; 207,208]

uridine (26/45); AZT (0.5 idoxuridine; thymidine;mM); dideoxycytidine (0.5 cytidine; uridinemM)

CNT2 human adenosine (8 uM); uridine formycin B, guanosine, [154,198]nucleoside/ (40/80 uM); inosine (5 adenosine, uridine, inosineNa+ symport uM)

Cnt2 rat [18,162]adenosine (6 uM); inosine formycin B, guanosine,(20 uM); uridine (20 uM); uridine, inosine, thymidinecladribine, thymidine (13uM)

PEPT1 human peptide/ H + glycylsarcosine (1.1 mM); ampicillin (50 mM); [4,31,43-45,PepTI rat symport ALA (0.28 mM); amoxicillin (13 mM); 55,120,180,PepTI rabbit ceftibuten (0.3 mM); cyclacillin (0.17 mM); 182,183,200]

cyclacillin (0.5 mM); cephalexin (5 mM); cefadroxilcephalexin; cefixime; (2 mM); cephradine (9 mM);cefadroxil; L-Val-ACV (5 cefdinir (12 mM); ceftibutenmM); L-Val-AZT; L-dopa-L- (0.6 mM); cefixime (7 mM);Phe; /brmy/-Met-Leu-Phe bestatin (0.5 mM); enalapril

(4.3 mM); captopril (9 mM); L-Val-ACV (0.74 mM)

PEPT2 human peptide/ H + glycylsarcosine (0.11 ampicillin (0.67 mM); [31,43-45,183]PepT2 rat symport mM); ALA (0.23 mM); amoxicillin (0.18 mM);PepT2 rabbit cephalexin; bestatin; L- cyclacillin (27 >wM); cephalexin

Val-ACV (50 juM); cefadroxil (3 juM);cephradine (47 uM); cefdinir(20 mM); ceftibuten (1 mM);cefixime (12 mM); bestatin (20uM); L-Val-ACV (0.39 mM)

^Abbreviations: PAH: p-amminohippurate; OA: organic anion; a-KG: a-ketoglutarate; MTX: methotrexate; PGE2, prostaglandin E2; BSP: bromosulfophthalein; DIDS: 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid; LTC4: leukotriene C4; LTD4 : leukotriene D4; CsA: cyclosporin A; E217£G: estradiol-17£-D-glucuronide; DNP-SG: dinitrophenyl-glutathione; GSSG, oxidized glutathione; GSH: reduced glutathione; AFB1-SG, 5- (aflatoxin B 1)-glutathione; E3040-glucuronide: 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole glucuronide; PMEA: 9-(2- phosphonylmethoxyethyl)adenine; AZTMP: azidothymidine monophosphate; CMFDA: 5-chloromethylfluorescein; FDA: fluorescein-diacetate; BCECF: 2', 7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein acetoxymethyl ester; TC: taurocholate; AZT: 3'-azido-3'-deoxythymidine; ALA: delta-aminolevulinic acid; L-Val-ACV: valacyclovir; PGA2-SG: 5-(prostaglandin A2)-glutathione; GS-DOX: 5-(doxorubicin)-glutathione; GS-DAU: 5- (daunorubicin)-glutathione; BQ123: cyclo-(Trp-Asp-Pro-Val-Leu); BQ485: perhydroazepino-N-carbonyl-(Leu-Trp-Trp); BQ518: cyclo-(Trp-Asp-Pro- Thg-Leu).

bSubstrates mentioned for some transporters are indicative but not complete.

19

Chapter One

interstitium lumen

K+

Na+

dicarboxylates

OA-

i nOAT1

n r ,

Na+/K+-ATPase Na+

SDCT2

Z ^ N a -

a-KG2

o a - — o

OA- ? SO 4SAT1

HCO3

oOA'

OATP1GSHo

OAT-K1/K2z n r1/K2z n

MRP2

CNT1/2r z i

OA-

*• OA-

OA-

Na+

nucleosides

PEPT1/2" o H+■4----- — H e H tid e s

Fig. 1. Schematic model of organic anion transporters in renal proximal tubule. Uptake of organic anions (OA-) across the basolateral membrane is mediated by the classical sodium-dependent organic anion transport system, which includes a-ketoglutarate (a-KG2-)/OA- exchange via the organic anion transporter (OAT1) and sodium-ketoglutarate cotransport via the Na+/dicarboxylate cotransporter (SDCT2). A second sodium-independent uptake system for bulky OA- has recently been identified, but its molecular identity is unknown. Intracellular accumulation occurs for substrates of both transport systems in mitochondria and vesicles of unknown origin. The apical (brush-border) membrane contains various transport systems for efflux of OA- into the lumen or reabsorption from lumen into the cell. The multidrug resistance transporter, MRP2, mediates primary active luminal secretion. The organic anion transporting polypetide, OATP1, and the kidney-specific OAT-K1 and OAT-K2 might mediate facilitated OA- efflux but could also be involved in reabsorption via an exchange mechanism. PEPT1 and PEPT2 mediate luminal uptake of peptide drugs, whereas CNT1 and CNT2 are involved in reabsorption of nucleosides.

20


Basolateral transport systems

Transport studies using isolated membrane vesicles and intact proximal tubules have identified and characterized the classical transport system for uptake of small organic anions across the basolateral membrane by using p-aminohippurate (PAH) or fluorescein as a model substrate [147]. These studies have established that uptake of PAH is a tertiary active process by indirect coupling to the Na+ gradient. This gradient, which is maintained by the Na+/K+ ATPase, drives Na+/dicarboxylate cotransport into the cell and enables uptake of PAH in exchange for a dicarboxylate ion. The PAH/dicarboxylate exchanger (Oat1) has been cloned from various species. Based on similarities with transport characteristics found in membrane vesicles, SDCT2 has been proposed as the

basolateral Na+/dicarboxylate cotransporter [19]. a-Ketoglutarate is by far the most abundant potential dicarboxylate counterion within the proximal tubular cell, and it has been shown that PAH or fluorescein uptake in renal proximal tubules increases with increasing internal a-ketoglutarate concentration [17,146,199]. The activity of the Na+/dicarboxylate exchanger accounts for approximately 60% of organic anion uptake [199], whereas the remainder can be explained by intracellularly stored a-ketoglutarate [146]. Mitochondrial metabolism seems responsible for the generation of this dicarboxylate, and from liver studies it is

known that the mitochondrial a-ketoglutarate concentration exceeds that in cytoplasm 3-6 times [170,173]. These findings imply that alterations in cellular metabolism may have a direct effect on the secretory function of kidney proximal tubules. Apart from the classical PAH transporter, an additional uptake system has been characterized by using the bulky organic anion fluorescein-methotrexate as a substrate [109]. Uptake of fluorescein-methotrexate is independent of Na+ and is not inhibited by PAH or the dicarboxylate ion glutarate. The molecular identity of this transporter, however, has not yet been established. Finally, a sulfate/anion exchanger has been characterized in basolateral membrane vesicles and cloned from rat [53,73,86,148]. This transporter exchanges sulfate for HCO 3" or oxalate in

21

Chapter One

a Na+-independent manner. Whereas multiple substrates have been proposed for the PAH/dicarboxylate and the sulfate/anion exchanger based on inhibition experiments [189], there is yet no evidence that these compounds are substrates themselves.

Organic anion transporter Oat1 (PAH/dicarboxylate exchanger)Various groups have cloned a PAH/dicarboxylate exchanger (Oat1) from rat and flounder by expression cloning in Xenopus laevis oocytes [166,176,191,205]. Furthermore, rat Oat1 sequences have enabled cloning of the human ortholog, OAT1 (86% identity to Oat1) [59,105,150,152]. Expression of rat Oat1 in Xenopus oocytes and human OAT1 in HeLa cells results in uptake of PAH, which is trans

stimulated by glutarate and cis-inhibited by glutarate, a-ketoglutarate, probenecid and fluorescein [166,176,191,205]. These transport characteristics correspond well to those established for PAH in basolateral membrane vesicles [147]. The localization to the basolateral membrane of proximal tubules further supports that OAT1 and Oat1 represent the classical basolateral PAH/dicarboxylate exchanger [59,186]. Oat1 has a broad substrate specificity and Oat1-mediated PAH transport is inhibited by various nonsteroidal anti-inflammatory drugs confirming previous results on in situ perfused rat proximal tubules using PAH as a substrate [3,166,187,189]. The substrate specificity of human OAT1 is more narrow, since it does not transport methotrexate or prostaglandin E2 unlike rat Oat1 [105]. Transport of PAH by both Oat1 and OAT1 is inhibited by phorbol 12-myristate 13- acetate (PMA) and this inhibition is reversed by staurosporin, indicating that organic anion uptake is negatively correlated to protein kinase C (PKC) activity [105,191]. These results are in agreement with previous reports on PKC-mediated inhibition of organic anion uptake into the opossum kidney cell line OK and proximal tubules of rabbit, killifish and flounder by using PAH, fluorescein, or dichlorophenoxyacetate as a substrate [46,54,121,178]. Since the Na+/K+ ATPase, the putative basolateral Na+/dicarboxylate exchanger SDCT2, as well as

22


Oat1/OAT1 have multiple PKC phosphorylation sites, it remains to be established whether PKC controls basolateral organic anion transport directly or indirectly.

Oat2 and Oat3/OAT3Functional expression in Xenopus oocytes of a putative rat liver transporter,

initially designated as NLT, revealed uptake of a-ketoglutarate, methotrexate, prostaglandin E2, acetylsalicylate, and PAH similar to rat Oat1 [165,172]. Since rat NLT also shows highest identity to rat Oat1 (42%), NLT has been renamed to Oat2 [165]. In addition to liver, Oat2 is also expressed in kidney. Kusahara et al. have cloned a novel transporter (Oat3), which shares an identity of 49% and 39% with Oat1 and Oat2, respectively [88]. Oat3 is expressed in brain, kidney, liver and eye, and also mediates uptake of PAH and other organic anions. In contrast to Oat1, uptake of organic anions mediated by Oat2 and Oat3 is independent of Na+ and glutarate [3,88,165]. This indicates that the presence of a concentration gradient is sufficient for Oat2 and Oat3 to enable organic anion transport. It is at present not clear whether Oat2 and Oat3 mediate efflux and/or reabsorption of organic anions because membrane localization and nephron distribution are not yet established.

Race et al. recently reported the cloning of OAT1 and a kidney-specific homolog called OAT3 (84% identity to Oat3) [150]. Expression of OAT3 in Xenopus oocytes, however, did not result in uptake of PAH [150], suggesting that OAT3 is not the human ortholog of rat Oat3. Brady et al. have cloned a murine gene encoding a putative transporter (Roct) with highest identity to Oat3 (92%) and OAT3 (83%) [13]. Expression of murine Oat3/Roct is abundant in kidneys of wildtype mice but markedly reduced in kidneys of mice homozygous for the recessive osteosclerosis (oc) mutation [13]. Both the oc mutation as well as the murine oat3/roct gene have been mapped to chromosome 19, although no mutations have yet been identified in the oat3/roct gene of oc mice [13]. In a similar way, the OAT3 gene has been mapped closely to the region were human recessive osteopetrosis, a disease with a similar pathophysiology as osteosclerosis,

23

Chapter One

has been mapped [13,56]. Further studies are required to elucidate the relation between these transporters and the phenotype of osteosclerosis and osteopetrosis.

Sulfate/anion exchanger Sat1Expression cloning using Xenopus laevis oocytes has identified a sulfate/anion exchanger (Sat1) from rat kidney and liver [9,73]. In the kidney, Sat1 is located at the basolateral membrane of proximal tubules [73]. Expression of Sat1 in Sf9 cells results in uptake of sulfate and oxalate, which is cis-inhibited by oxalate and sulfate, respectively [73]. In addition, DIDS but not succinate inhibits Sat1- mediated sulfate uptake [9]. These transport characteristics are in close agreement to those found in proximal tubule basolateral membrane vesicles [53,86,148]. The cloning of Sat1 enables to answer the question whether the compounds proposed as substrates based on earlier inhibition studies are indeed transported by Sat1.

Multidrug resistance protein MRP1The human multidrug resistance protein 1 (MRP1) is a member of the large family of ATP-binding cassette (ABC) proteins and functions as an ATP-dependent transporter of anionic conjugates, such as leukotriene C4, S-(dinitrophenyl)- glutathione, estradiol-17£-D-glucuronide, and etoposide-glucuronide [93,98, 99,132]. MRP1 is expressed in various tissues, including blood cells [39,213]. Mice homozygous for a disrupted mrp1 gene (mrp1 -/-) have an impaired response to an inflammatory stimulus, probably as a consequence of decreased leukotriene C4 secretion from leukocytes [201]. Murine Mrp1 is localized to the basolateral membrane of various epithelial cells and human MRP1 is routed to the basolateral membrane when expressed in the epithelial cell lines LLC-PK1 and MDCKII [33,35,203]. In murine kidney, Mrp1 is expressed at the basolateral membrane of cells of Henle's loop and the cortical collecting duct [144,203]. Although previously suggested [155], Mrp1 does not appear to be expressed in the proximal tubule [144,203].

24


Human MRP1 is frequently overexpressed in various multidrug resistant (MDR) cancer cell lines selected with cationic (chemotherapeutic) drugs [21]. Transfection of a human MRP1 cDNA into drug-sensitive cells results in resistance to various cationic drugs, such as vincristine, doxorubicin, and etoposide [22,50,211]. In addition, mrp1 -/- mice are hypersensitive to such drugs [104,201,203]. Since administration of etoposide to mrp1 -/- mice results in polyurea, MRP1 might protect distal parts of the nephron, which are exposed to high concentrations of drugs as a result of water reabsorption [203].

MRP3, MRP4, MRP5 and MRP6Searching of the GenBank database EST sequences enabled identification of four additional human MRP-like genes alongside MRP1 and MRP2 (see apical organic anion transport systems). Expression of MRP3 [5,78,82,85,188], MRP4 [82,164], MRP5 [5,82,118], and MRP6 [83] mRNA has been found in various tissues, including kidney. However, Lee et al. [90] did not find expression of MRP4 in the kidney.

The subcellular localization and nephron distribution of these novel MRPs in the kidney is at present unknown. However, immunohistochemistry on liver sections has demonstrated the presence of human MRP3 at the basolateral membrane of intrahepatic bile-duct epithelial cells (cholangiocytes) and hepatocytes, whereas rat Mrp6 has been located specifically to the hepatocyte lateral membrane [84,85,107]. Similarly, human MRP5 is confined to the basolateral membrane of various epithelial cells [202]. Transport characteristics of several of these novel MRPs have been reported recently. MDCKII cells overexpressing human MRP3 show increased efflux of S-(dinitrophenyl)- glutathione across the basolateral membrane compared to the parental cells [84]. Furthermore, MRP3 confers resistance to the chemotherapeutic drugs etoposide, teniposide, and methotrexate [84]. In the cell line HepG2, endogenously expressed MRP3 mRNA is induced by phenobarbital [78]. Membrane vesicles from LLC-PK1 cells transfected with a rat mrp3 cDNA exhibit ATP-dependent

25

Chapter One

uptake of estradiol-175-D-glucuronide and E3040-glucuronide, but not leukotriene C4 or S-(dinitrophenyl)-glutathione [58]. In a T-lymphoid cell line, overexpression of human MRP4 mRNA and MRP4 protein was found to be correlated with enhanced ATP-dependent efflux of nucleoside monophosphate analogs [164]. Expression in HEK293 cells of human MRP5 conjugated to green fluorescent protein results in reduced labeling of some fluorescent anionic agents [118]. In a preliminary report, rat Mrp6 was shown to mediate ATP-dependent transport of the anionic cyclopentapeptide endothelin antagonist BQ123 [107]. Further studies are required to determine the transport characteristics of these MRPs and to define their role in the kidney.

Intracellular disposition of anionic drugs

Anionic drugs accumulate in proximal tubules as a result of secretory transport, possibly in combination with reabsorption. The degree of accumulation is determined by the extent at which anionic drugs are actively transported across the basolateral membrane, their intracellular disposition and ease at which they can be transported across the brush-border membrane into the tubular lumen.

In vitro studies in isolated proximal tubular cells have indicated that, at low medium concentrations, PAH and fluorescein accumulate intracellularly at concentrations 3-5 times the medium concentration [112]. Fluorescein accumulation in proximal tubular cells during secretory transport has recently been confirmed in rat kidney in vivo [1 2 ]. Confocal microscopic images of rat proximal tubular cells showed compartmentation of fluorescein in subcellular organelles. In particular mitochondria appear to concentrate fluorescein via the metabolite anion transporters in the inner membrane [110,112,184]. On the other hand, Miller et al. suggested nocodazole-sensitive vesicular compartmentation in crab urinary bladder [122,124,125]. Nocodazole disrupts the Golgi apparatus and subsequently microtubules, which are involved in the movement of transporting

26


vesicles thorough the cells [14]. This indicates that at least two different compartments may be involved in the intracellular sequestration of organic anions. The involvement of microtubules in vesicular compartmentation suggests a role in transcellular transport and/or secretion. Another possibility is that endosomal membranes play a role in the rapid and directed trafficking of transporters to and from the apical membrane.

Hardly any research is done on transcellular transport of organic anions in the renal proximal tubule. Two decades ago the presence of anion binding proteins were shown, of which ligandin or glutathione S-transferase B is the most important [16,96,167]. Several anionic drugs interact with these binding proteins, suggesting that they play a role in the reduction of free cytoplasmic drug concentrations and maybe in transcellular drug trafficking [49,77,96]. Binding to glutathione S- transferase, however, appeared not to be a determinant for the rate of luminal secretion [143]. Unfortunately, more recent data on the role of binding proteins is not available.

Finally, metabolism may be an important intracellular event associated with renal anionic xenobiotic disposition. The biotransformation pathways reported involve oxidative, reductive and hydrolytic reactions (phase I reactions), and conjugation to glucuronide, sulfate or reduced glutathione (phase II reactions) [48,97,102,129]. Microsomal oxidation through cytochrome P450-dependent mechanisms takes place predominantly in kidney proximal tubules [102]. Glucuronide conjugates are formed through the enzymatic activity of UDP- glucuronosyltransferase, of which various isozymes have been identified in the kidney. Renal sulfate conjugation is catalyzed by sulfotransferase, but is quantitatively less than glucuronide conjugation [97,102]. Conjugation to reduced glutathione (GSH) is catalyzed by GSH-transferase, and the conjugates may subsequently be transformed into cysteine conjugates and mercapturic acid by y- glutamyltranspeptidase and N-acetyltransferase, respectively [23]. The enzymes involved in glucuronide, sulfate or GSH conjugation are not uniformly distributed in the kidney, but show the highest activity in the proximal tubule

27

Chapter One

[62,97,102,209]. The different enzymes involved in GSH-derived biotransformation reactions (y-glutamyltranspeptidase and N-acetyltransferase) express a high activity predominantly in S3 segments of the proximal tubule [61]. Biotransformation reactions are primarily detoxification mechanisms, although toxic metabolites may be produced as well (e.g. acetaminophen [29,133], halogenated alkanes [133], cisplatin [26,168], cyclosporin A [7], ochratoxin A [30,47]).

Apical transport systems

Much of the transport systems for organic anions in the brush-border membrane of the proximal tubule have initially been characterized in membrane vesicle studies [147]. Small organic anions, such as PAH and fluorescein, are excreted via a facilitated transporter driven by the potential difference of about -70 mV from cell to lumen. In addition, an anion exchange mechanism has been identied in some species, such as dog, rat and human but not in rabbit. Neither of these efflux systems have been cloned. Based on inhibition experiments in in situ perfused proximal tubules, Ullrich et al. suggested multiple substrates for these organic anion transporters [190]. Studies with intact killifish proximal tubules have indicated two efflux pathways for organic anions [108,109,123]. One pathway mediates Na+-dependent efflux of small organic anions, independent of the membrane potential [109,123]. Bulky organic anions, such as fluorescein- methotrexate and lucifer yellow, are excreted via an energy-dependent efflux system, which is insensitive to PAH and depletion of Na+' but inhibited by leukotriene C4 and S-(dinitrophenyl)-glutathione [108,109]. A likely candidate for this transport system is the apical ATP-dependent anionic conjugate transporter Mrp2. Apart from efflux mechanisms, the brush border membrane also contains transport systems for reabsorption of compounds from primary urine. Small anionic peptides are actively taken up via H+/peptide cotransporters by coupling

28


to the H+-gradient [27]. In addition, anionic nucleosides are actively taken up by Na+/nucleoside cotransporters by coupling to the Na+-gradient [40,51,204].

MRP2The multidrug resistance protein 2 (MRP2) is an ATP-dependent organic anion transporter with highest identity to MRP1 (48%) and MRP3 (46%). MRP2, also described as canalicular multispecific organic anion transporter (cMOAT), is expressed at the apical membrane of hepatocytes [15,140], renal proximal tubules [163], and small intestinal villi (see chapter 3). In liver, MRP2 plays an important role in the biliary excretion of multiple conjugated and unconjugated organic anions across the canalicular (apical) membrane. Defective function of Mrp2, as observed in the two mutant rat strains EHBR and GY/TR", results in hyperbilirubinemia [15,66,140]. Furthermore, in patients with Dubin-Johnson syndrome, a rare autosomal recessive liver disorder with a similar phenotype as the mutant rats, mutations have been identified in the MRP2 gene [74,141,196].

The transport characteristics of Mrp2 have been investigated extensively in comparative studies with wildtype and Mrp2-deficient rats using perfused liver, and isolated canalicular membrane vesicles or hepatocytes [136,175]. In contrast to liver canalicular membrane vesicles, membrane vesicles from the proximal tubule brush-border membrane are unsuitable for characterizing Mrp2-mediated transport. Since these vesicles are exclusively orientated rightside-out, the ATP- binding site is inaccessible for extravesicular ATP [52]. Studies with killifish renal proximal tubules have indicated the presence of an MRP-like transporter involved in the energy-dependent and leukotriene C4-sensitive efflux of fluorescein- methotrexate and lucifer yellow [108,109]. Immunohistochemistry with a polyclonal antibody raised against rabbit Mrp2 has located a killifish ortholog to the brush-border membrane, which further supports this hypothesis [111]. Functional evidence exists for the presence of apical transporters other than Mrp2 involved in renal excretion of organic anions. For example, efflux of lucifer yellow from killifish proximal tubules is only partly inhibited by the Mrp2 substrates

29

Chapter One

leukotriene C4 and S-(dinitrophenyl)-glutathione [108]. Also, renal clearance of the

Mrp2 substrates a-naphtyl-^-D-glucuronide [28], E3040-glucuronide [179], cefpiramide [130], the quinolone HSR-903 [131], and lucifer yellow (R. Masereeuw and F.G.M. Russel, unpublished) is not impaired in Mrp2-deficient rats.

Like MRP1 and MRP3, human MRP2 not only transports anionic conjugates but also confers resistance to various cationic chemotherapeutic drugs [24,81]. Studies with membrane vesicles from cells expressing human MRP1 have indicated that uptake of cationic drugs, such as vincristine, daunorubicin, and aflatoxin B1 only occurs in the presence of physiological concentrations of GSH [69,99-101]. A similar GSH dependency of ATP-dependent vinblastine transport has been shown for rabbit Mrp2 (see chapter 4). This explains the finding that MRP1- and MRP2-mediated drug resistance is reversed by an inhibitor of GSH synthesis [24,195,212]. For human MRP1, the mechanism involved in GSH- stimulated ATP-dependent transport of vincristine has been identified as a GSH/vincristine cotransport mechanism [100]. Daunorubicin and etoposide, unlike vincristine, did not stimulate GSH transport indicating the involvement of a mechanism different from cotransport [100]. Recent studies have shown that MRP2 [142], like MRP1 [142,212] and MRP5 [202] but not MRP3 [84], transports GSH itself.

Regulation of Mrp2-mediated transport has been investigated in isolated rat hepatocytes and killifish renal proximal tubules, however, results in these tissues are at variance. Both cAMP and PKC stimulated Mrp2-mediated efflux of an anionic conjugate across the hepatocyte apical membrane [156,157]. At least for the effect of cAMP, it has been shown to be a result of stimulated sorting of Mrp2 containing vesicles to the apical membrane [157]. In the killifish proximal tubule, efflux of fluorescein-methotrexate is negatively correlated with PKC activity [111]. Activation of the signal transduction pathway occurs by binding of endothelin-1 to the B-type receptor [111]. Since endothelins are involved in many processes in the kidney, this suggests a specific regulatory pathway for renal Mrp2.

30


Organic anion transport polypeptides Oatp1 and Oatp3The organic anion-transporting polypeptide 1 (Oatp1) is a Na+- and ATP- independent transporter originally cloned from rat liver [6 8 ]. Oatp1 is localized to the basolateral membrane of hepatocytes where it plays a major role in uptake of a variety of anionic, neutral and cationic compounds from the blood [119]. In contrast, Oatp1 is located at the apical membrane of S3 proximal tubules [8 ]. Studies with transiently transfected HeLa cells have indicated that Oatp1 mediates uptake of taurocholate in exchange for HCO 3" [161]. In addition, uptake of taurocholate by Oatp1 expressed in Xenopus oocytes is accompanied by efflux of GSH [94]. As an anion/GSH exchanger, Oatp1 might mediate reabsorption of organic anions rather than efflux in the kidney. In this respect, reabsorption of ochratoxin A in proximal tubules is inhibited by bromosulfophthalein, which suggests the involvement of Oatp1 as both compounds have been identified as substrates [25,32]. On the other hand, since Oatp1 transports various anionic conjugates it might function as an efflux transporter, representing the transport mechanism maintained in kidneys of Mrp2-deficient rats. Abe et al. reported the cloning of an Oatp1-homolog, called Oatp3 (80 and 82% identity to Oatp1 and Oatp2, respectively) [1]. Oatp3 is highly expressed in kidney and mediates transport of thyroid hormones and taurocholate like Oatp1 and Oatp2 [1,41]. However, no data are yet available on the nephron distribution and membrane localization of Oatp3.

Organic anion transporters Oat-k1 and Oat-k2Oat-k1 and Oat-k2 are kidney-specific Na+- and ATP-independent organic anion transporters with highest identity to Oatp1 (72% and 65%, respectively) [114,158]. Both transporters are confined to the brush-border membrane of proximal tubular cells [114,117]. Oat-k1 and Oat-k2 mediate transport of methotrexate and folate, whereas Oat-k2 also transports prostaglandin E2 and taurocholate [114,158]. Additional studies are required to elucidate whether Oat-k1 and Oat-k2 mediate efflux or reabsorption of organic anions. However, Oat-k1 expressed in MDCK

31

Chapter One

cells transports methotrexate across the apical membrane in both directions [115]. Similarly, Oat-k2 transports taurocholate bidirectionally across the apical membrane upon expression in MDCK cells [114]. In a recent study, Oat-k1 was proposed as the ochratoxin A reabsorption pathway in proximal tubules based on the inhibitory effect of bromosulfophthalein [25]. Whereas bromosulfophthalein indeed inhibits Oat-k1 mediated transport [115,158], it also inhibits Oat-k2 mediated transport [114]. Furthermore, there is no evidence to suggest that ochratoxin A is a substrate of either of these transporters. In this respect, various organic anions (i.e. nonsteroidal anti-inflammatory drugs, PAH, digoxin, probenecid), bile acid analogs, and steroids are potent inhibitors but no substrates of Oat-k1 and Oat-k2 [114-116,158]. Several of these drugs can accumulate to high concentrations in the proximal tubule, suggesting that under these conditions both transporters are inhibited.

Na+/nucleoside cotransporters CNT1 and CNT2The concentrative nucleoside transporters CNT1 and CNT2 are involved in Na+- dependent transport of endogenous nucleosides and various synthetic (anionic) nucleosides, which are of clinical importance for their use in the treatment of cancer and viral infections [139]. CNT1 (N2 subtype or cit) is selective for pyrimidines whereas CNT2 (N1 subtype or cif) favors transport of purines, although uridine and adenosine are transported by both proteins [18,36,60,153,154,162,198,207,208]. Structural features markedly distinguishes rat Cnt2 from human CNT2, moreover, Cnt2 transports thymidine in contrast to CNT2 [18,154,198]. Northern blotting and reverse transcriptase polymerase chain reaction have identified expression of human CNT1 and CNT2 mRNA in the kidney [153,154,198]. Although their membrane localization has not been established by immunohistochemistry, Na+/nucleoside cotransport into cells overexpressing CNT1 or CNT2 resemble transport characteristics found in brush- border membrane vesicles [40,51,204]. This indicates that CNT1 and CNT2

32


mediate Na+/nucleoside cotransport across the brush-border membrane into the proximal tubule.

H+/peptide cotransporters PEPT1 and PEPT2Peptide transporters are involved in H+-dependent transport of small peptides and various peptide-like compounds such as anticancer drugs (bestatin, delta- aminovulinic acid), prodrugs (L-dopa-L-Phe, L-Val-AZT), inhibitors of angiotensin converting enzyme ACE (captopril, enalapril), and various anionic S-lactam antibiotics such as cephalosporins (cephalexin, cepharadine, cefadroxil, cefdinir) and penicillins (cyclacillin, ampicillin) [27,91]. Two peptide transporters have been cloned, i.e. a low-affinity transporter from intestine (PEPT1) and a high- affinity transporter from kidney (PEPT2) [27,91]. Immunohistochemistry has located PEPT2 to the proximal tubule brush-border membrane and characteristics of H+/peptide cotransport into various cell types expressing PEPT2 resemble transport characteristics found in brush-border membrane vesicles [10,127,128,151,169,171,177,185]. This indicates that PEPT2 mediates H+/peptide cotransport from the primary urine into the proximal tubule [27,169]. PEPT1 has also been located to the brush-border membrane but is difficult to characterize in membrane vesicles due to its relatively low expression and the interference of substrates with PEPT2 [27]. However, the ACE inhibitors enalapril and captopril are not transported by PEPT2, and these drugs have recently been used to characterize PEPT1-mediated transport in kidney [182].

Conclusive remarks

The past few years have witnessed great advances in our understanding of the molecular pharmacology of renal organic anion transport. Considerable progress has been made in cloning key proteins involved in the transport of anionic xenobiotics and metabolites and the list of cloned organic anion transporters is steadily growing. The challenge of future work will be to integrate this information

33

Chapter One

with (patho)physiological, pharmacological and toxicological investigations at the cellular and organ level. There is still a large gap in our knowledge about the relative contribution of individual transporters to the membrane steps involved in tubular secretion of specific anionic substrates. More detailed knowledge of the cloned carrier proteins in expression systems and the availability of specific antibodies will allow more fundamental insight into membrane translocation at the molecular level, as well as participation in overall transepithelial secretion. This will also facilitate the research on the interaction of cellular messengers with the carrier proteins and the way these transporters are synthesized and targeted to specific membrane sites in the normal and diseased kidney. An important approach to study the in vivo function of transporters and their mutual interaction is to develop and characterize (multiple) null mutants of the genes encoding these proteins. Finally, it has become increasingly clear that intracellular disposition is an important step in the active secretion of anionic xenobiotics. How these processes interact with the membrane transport mechanisms to produce secretion and at what level and to what extent they are regulated will be important questions to answer.A detailed knowledge of the renal mechanisms that govern intracellular distribution and membrane transport of xenobiotics in the kidney, is essential for the development of clinically useful drugs and will advance our understanding of the molecular, cellular and clinical basis of renal drug clearance, drug-drug interactions, drug targeting to the kidney and xenobiotic-induced nephropathy.

Aim and Outline of this Thesis

In 1996, at the department of Cell Physiology (University of Nijmegen), a novel homolog of MRP1 was cloned from a rabbit ileum cDNA library (van Kuijck et al. 1996, Proc. Natl. Acad. Sci. 93, 5401). At the same time, Mrp2 was cloned from rat by three collaborative groups in Amsterdam (Paulusma et al., 1996, Science 271, 1126). Comparison of the tissue distribution and the nucleotide sequence

34


suggested that our clone is the rabbit ortholog of rat Mrp2. In this thesis, we functionally expressed rabbit Mrp2 in Sf9 cells to characterize its transport properties and to use this model system to identify putative substrates, with emphasis on compounds that are excreted by the kidney. In chapter 2, we describe the expression of rabbit Mrp2 in Spodoptera frugiperda (Sf9) cells and show Mrp2-mediated uptake of tracer substrates in isolated membrane vesicles. We investigated the immunolocalization of Mrp2 in rabbit small intestine, liver and kidney (chapter 3). In chapter 4, the mechanisms and interaction of vinblastine and GSH transport by Mrp2 were studied. In chapter 5, we investigated whether Mrp2 transports PAH in view of its putative role as renal organic anion transporter. In chapter 6, the results are summarized and a general discussion with future perspectives is given.

References

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44


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45

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46

Chapter 2

Adenosine Triphosphate-Dependent Transport of Anionic Conjugates by the Rabbit Multidrug Resistance Protein Mrp2 Expressed in Insect Cells

Remon A.M.H. van Aubel1, Marcel A. van Kuijck3, Jan B. Koenderink2,Peter M.T. Deen3, Carel H. van Os3, and Frans G.M. Russel1

Depts. o f1Pharmacology and Toxicology, 2Biochemistry, and 3Cell Physiology, University of Nijmegen, The Netherlands

Molecular Pharmacology, 1998, volume 53, pages 1062-1067 (adapted version)

47

Functional Expression of Rabbit Mrp2 in Insect Cells

Summary

The multidrug resistance protein Mrp2 is expressed in liver, kidney and small intestine and mediates ATP-dependent transport of conjugated organic anions across the apical membrane of epithelial cells. We recently cloned a rabbit cDNA encoding a protein that on basis of highest amino acid homology and similar tissue distribution was considered to be the rabbit homolog of rat Mrp2. To investigate whether rabbit Mrp2 mediates ATP-dependent transport similar to rat Mrp2, we expressed rabbit Mrp2 in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. Mrp2 was expressed as an underglycosylated protein in Sf9 cells and to a higher level compared with rabbit liver and renal proximal tubules. Both 17ß-estradiol-17-ß-D-glucuronide ([3H]E2i 7 ßG, 50 nM) and [3H]leukotriene C4 (3 nM) were taken up by Sf9-Mrp2 membrane vesicles in an ATP-dependent fashion. Uptake of [3H]E2i 7 ßG was dependent on the osmolarity of the medium and saturable for ATP (Km = 623 ^M). Leukotriene C4, MK571, phenolphthalein glucuronide, and fluorescein-methotrexate were good inhibitors of [3H]E2i7ßG transport. The inhibitory potency of cyclosporin A and methotrexate was

moderate, whereas fluorescein, a-naphthyl-ß-D-glucuronide and p-nitrophenyl-ß- D-glucuronide did not inhibit transport. In conclusion, we show direct ATP- dependent transport by recombinant rabbit Mrp2 and provide new data on Mrp2 inhibitor specificity.

(available online at http://molpharm.aspetjournals.org)

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http://molpharm.aspetjournals.org

Chapter Two

Introduction

Elimination of endogenous waste products and xenobiotics from the body is mediated by renal and hepatic transport pathways. Excretion of anionic conjugates across liver canalicular (apical) membranes into bile is mediated by the multidrug resistance protein MRP2 [26]. Initially, this transporter was named cMOAT and characterized by using natural mutant strains of Wistar (TR-) and Sprague-Dawley (EHBR) rats [26]. Recently, cloning of rat Mrp2 revealed that the impaired conjugate transport in canalicular membranes of these rats is caused by a premature termination of the mrp2 gene product [11,27]. Similarly, a mutation leading to a truncated MRP2 was identified in a patient with Dubin-Johnson syndrome, a disease that resembles the TR- phenotype [28].

Database analysis revealed that rat Mrp2 is strongly related to the human multidrug resistance protein MRP1, a member of the superfamily of ABC proteins [3,11,27]. Originally, MRP1 was identified due to its overexpression in a multidrug resistant cell line and its ability to confer resistance to chemotherapeutic drugs [21]. Using isolated membrane vesicles from MRP7-transfected cells, it has been shown that MRP1 is also capable of transporting anionic conjugates in an ATP- dependent manner. Although MRP1 and MRP2 share substrate specificity, these transporters show differences in their tissue distribution. MRP1 is expressed, predominantly intracellularly, in numerous tissues such as lung, heart, and kidney [7]. In contrast, MRP2 was detected in small intestine and apical (canalicular) membranes of hepatocytes and cells of renal proximal tubules [3,27,33].

W e cloned a rabbit cDNA encoding an ABC-transporter that on basis of similar tissue distribution and highest amino acid homology was considered to be the rabbit homolog of rat Mrp2 [37,38]. On injection of its cRNAs in Xenopus laevis oocytes, we observed in a few cases a cAMP-dependent chloride conductance [38]. To investigate whether rabbit Mrp2 functions as an ATP-dependent organic anion transporter similar to rat Mrp2, we expressed rabbit Mrp2 in Sf9 cells using recombinant baculovirus and studied uptake of the anionic conjugates E217SG and

50


LTC4 into isolated membrane vesicles. In addition, the effect of various inhibitors on Mrp2-mediated [3H]E217£G transport was investigated.

Experimental procedures

Materials. [14,15,19,20-3H]LTC4 (165 Ci/mmol) and [6,7-3H]E217GG (55 Ci/mmol) were purchased from NEN Life Science Products (Hoofddorp, The Netherlands). ATP, 5'-AMP,

LTC4 , E2 1 7 GG, methotrexate (MTX), cyclosporin A (CsA), a-naphthyl-G-D-glucuronide, phenolphthalein glucuronide, and p-nitrophenyl-G-D- glucuronide were purchased from Sigma (Zwijndrecht, The Netherlands). FL-MTX and FL were purchased from Molecular Probes (Leiden, The Netherlands). Creatine phosphate and creatine kinase were purchased from Boehringer Mannheim (Almere, The Netherlands). CELLFECTIN and competent DH10BAC Escherichia coli cells were purchased from Life Technologies (Breda, The Netherlands). PNGase F was purchased from New England Biolabs (Westburg, Leiden, The Netherlands). MK571 was a generous gift of Dr. A. W . FordHutchinson (Merck Frosst, Center for Therapeutic Research, Quebec, Canada).

Preparation of antibodies. Rabbit polyclonal antibodies were directed against two different epitopes of rabbit Mrp2 [38]. Antiserum k78mrp2 was obtained by immunizing rabbits with a glutathione S-transferase fusion protein containing the 159 carboxyl-terminal amino acids (1405-1564) of Mrp2. Antiserum k51mrp2 was obtained by immunizing rabbits with a synthetic peptide (FQKRQQKKSQKNSRLQGL) corresponding to amino acids 257-274 of Mrp2 coupled to keyhole limpet haemocyanin. Rabbits were immunized with 400 ^g of either the fusion protein or the synthetic peptide mixed with Freund's complete adjuvants. At 3-week intervals after priming, rabbits were boosted with 200 ^g of proteins supplemented with incomplete adjuvants. Test bleedings were checked for the presence of Mrp2-specific antibodies using enzyme-linked immunosorbent assay.

Expression construct. The vector pFASTBAC1 (Life Technologies) contains an expression cassette that consists of a polyhedrin promoter, a multiple cloning site and an SV40 poly(A)+ signal inserted between the left and right arms of the bacterial transposon Tn7. Cloning of a rabbit mrp2 cDNA into pFASTBAC1 was accomplished in two steps: (1) from

51

Chapter Two

the pBluescript KS+ construct pBSmrp2, which contains the entire rabbit mrp2 coding sequence (nucleotides 347-5038) [38], a 2.7 kb XbaI/PstI fragment (nucleotides 26905407) was cloned into the XbaI and PstI sites of the multiple cloning site of pFASTBAC1 to create pFASTBAC-m1; and (2) to minimize the 5'-untranslated region, the 5' coding sequence of rabbit Mrp2 was amplified by polymerase chain reaction using the forward primer Mrp2-F1 (5'-ATGCTGGATAAGTTCTGCAAC-3'; nucleotides 347-368), which contains the ATG start codon (underlined), and the reverse primer Mrp2-R1 (5'-GCAGGAGTAGGCCAGATTAG-3'; nucleotides 844-824). The resulting polymerase chain reaction product of 498 bp was cloned into the SmaI site of pBluescript KS + and its sequence was verified by dideoxy sequence analysis [32]. From this construct, a StyI/HincII fragment was removed and replaced by a StyI/EcoRV fragment (nucleotides 480-3007) from pBSmrp2. Next, a 2.5 kb BamHI fragment of this construct, containing the 5'-region of mrp2 (nucleotides 347-2868), was cloned into the BamHI site of pFASTBAC1-m1 and its orientation was determined. The selected construct, designated pFBmrp2, contains a full-length rabbit mrp2 cDNA with the ATG start codon immediately downstream of the polyhedrin promoter.

Production of recombinant baculovirus and viral infection. Baculovirus encoding rabbit Mrp2 was generated using the Bac-to-Bac baculovirus expressing-system (Life Technologies). Competent DH10BAC E. coli cells harboring a baculovirus shuttle vector (bacmid) with a Tn7 attachment site were transformed with the pFBmrp2 construct. On transposition between the Tn7 sites, recombinant bacmids were selected and isolated according to the manufacturer. Subsequently, insect Sf9 cells were transfected with recombinant bacmids using CELLFECTIN reagent. After 3 days, culture medium was collected and used to infect fresh Sf9 cells. Four days after infection, stocks of amplified virus were made. Sf9 cells (106/ ml) were grown as 100 ml suspension cultures and infected at a multiplicity of infection (MOI) of 1-5 with recombinant baculovirus encoding Mrp2. For control experiments, Sf9 cells were infected with recombinant baculovirus encoding G-glucuronidase (Life Technologies) or the G-subunit of H+/K+ ATPase [16]. Three days after infection, membrane fractions were isolated (see below).Isolation of membrane fractions. Crude membrane fractions and membrane vesicles from infected Sf9 cells were isolated as described by Leier et al. [19] with modifications. Briefly, cells were collected and resuspended in hypotonic buffer (0.5 mM sodium phosphate, 0.1

52


mM EDTA, pH 7.0) supplemented with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 5 ^g/ml aprotinin, 5 ^g/ml leupeptin, 1 mM pepstatin). Cells were stirred gently on ice for 90 min and the resulting lysate was centrifuged at 100,000 x g for 40 min at 4

°C. The pellet of crude membranes was resuspended in TS-buffer (10 mM Tris-Hepes, 250 mM sucrose pH 7.4) using a Potter homogenizer and the homogenate was centrifuged at

12,000 x g for 10 min at 4 °C. The postnuclear supernatant was centrifuged at 100,000 x g

for 40 min at 4 °C and the pellet obtained was resuspended in TS-buffer with a tight-fitting Dounce (type B) homogenizer. The suspension was layered over 38% sucrose in 5 mM

Hepes/KOH, pH 7.4, and centrifuged at 100,000 x g for 2 hr at 4 °C. The interphase was collected and homogenized on ice with a tight-fitting Dounce (type B) homogenizer and

the suspension was centrifuged at 100,000 x g for 40 min at 4 °C. The resulting pellet was resuspended in TS-buffer and passed through a 27-gauge needle for 30 times. Membrane

vesicles were frozen and stored at -80 °C until use. Sidedness of membrane vesicles was assessed by measuring 5'-nucleotidase activity [6], and it was determined that ~65% of the vesicles were orientated inside-out. Rabbit liver and kidney were excised and renal proximal tubular cells were isolated by immunodissection as described previously [30]. Crude membrane fractions were isolated as described previously [23]. Briefly, liver, kidney, and renal proximal tubular cells were homogenized in buffer A (300 mM sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2) supplemented with protease inhibitors as

described above. Homogenates were centrifuged at 500 x g for 15 min at 4 °C, followed

by centrifugation of the supernatant at 200,000 x g for 60 min at 4 °C. Subsequently, the pellet was resuspended in buffer A. For all membrane preparations, protein concentration was determined using the BioRad protein assay (BioRad Laboratories, Veenendaal, The Netherlands).

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Chapter Two

Deglycosylation studies and immunoblot analysis. Crude membrane fractions from Sf9 cells infected with Mrp2-encoding baculovirus and from rabbit kidney were treated with PNGase F according to the manufacturer. Protein-equivalents (see figure legends) were solubilized in Laemmli's sample buffer supplemented with 100 mM dithiothreitol, heated

for 10 min at 65 °C, subjected to SDS polyacrylamide gel electrophoresis, and transferred to Hybond-C pure nitrocellulose membrane (Amersham, Buckinghamshire, UK) as described previously [5]. Transfer of proteins was confirmed by the reversible staining of the membrane with Ponceau Red. Subsequently, the blot was blocked for 60 min with 5% nonfat dry milk powder in Tris-buffered saline supplemented with 0.3% Tween-20 (TBS-T) and washed twice with TBS-T. To detect rabbit Mrp2 proteins, the membrane was incubated overnight at 4 0C with antiserum k78mrp2 or k51mrp2 diluted 1:5000 in TBS-T. After two times washing for 5 min with TBS-T, the blot was blocked for 30 min as described above. The blot was then washed twice with TBS-T and incubated at room temperature for 60 min with affinity-purified horseradish peroxidase-conjugated goat antirabbit IgG (Sigma Immunochemicals, St. Louis MO) diluted 1:5000 in TBS-T. Finally, the blot was washed twice for 5 min with TBS-T and TBS, respectively. Proteins were visualized using enhanced chemiluminescence (Pierce, Rockford IL).

Transport studies in membrane vesicles. Uptake of [3H]LTC4 into membrane vesicles was measured by using a rapid filtration technique [19]. Briefly, membrane vesicles (20 ^g

protein-equivalent) were rapidly thawed and incubated at 37 °C in the presence of 4 mM MgATP, 10 mM MgCh, 10 mM creatine phosphate, 100 ^g/ml creatine kinase, and 3 nM [3H]LTC4 in a final volume of 120 ̂ l of TS-buffer (10 mM Tris-Hepes, 250 mM sucrose, pH7.4). At indicated times, 20-yl samples were taken from the reaction mixture, diluted in ice-cold TS-buffer and filtered through nitrocellulose filters (0.45 ^m pore size, Schleicher & Schuell, Dassel, Germany) using a filtration device (Millipore Corp., Bedford, MA). Filters were washed once with 5 ml TS-buffer and dissolved in liquid scintillation fluid to determine the bound radioactivity. In control experiments, 4 mM MgATP was replaced by 4 mM 5'-AMP. Net ATP-dependent transport was calculated by subtracting values in the presence of 5'-AMP from those in the presence of ATP. Uptake of [3H]E217GG at a final concentration of 50 nM was done similarly as described for [3H]LTC4, except that a 50 ^g protein-equivalent of membrane vesicles was used.

54


A200 —

116 — 97 —

66

B KIDNEY Sf9-Mrp2_ + _ + PNGase F

200

116— 97—

***4 —m

k51mrp2 k78mrp2

Fig. 1. Immunoblot analysis of Sf9 cells infected with recombinant baculovirus encoding Mrp2.A, Crude membrane fractions were isolated from Sf9 cells infected with a control baculovirus (Sf9-c) or recombinant baculovirus encoding Mrp2 (Sf9-Mrp2). Proteins (5 /jg) were separated on a 6% SDS polyacrylamide gel, transferred to a nitrocellulose membrane, and incubated with Mrp2 polyclonal antiserum k78mrp2 or k51mrp2. Proteins were visualized using enhanced chemiluminescence. Sizes of protein standards are indicated in kilodaltons. B, Protein-equivalents of crude membrane fractions from rabbit kidney (20 jg ) and from Sf9 cells infected with recombinant baculovirus encoding Mrp2 (5 jg ), were treated without (-) or with (+) PNGase F and subjected to immunoblot analysis using antiserum k78mrp2 as described in A.

Results

Sf9 insect cells were infected with recombinant baculovirus encoding rabbit Mrp2 or control baculovirus. Crude membranes were prepared and subjected to immunoblot analysis using antiserum k78mrp2 and k51mrp2. Both antisera detected a protein of ~180 kDa in membranes from cells infected with baculovirus encoding Mrp2 (Fig. 1A, Sf9-Mrp2) but not in membranes from cells infected with control baculovirus (Fig. 1A, Sf9-c). This size is smaller than cMoat/Mrp2 detected in liver and kidney, which has been reported to have a molecular weight of ~190 kDa [3,27,33]. To investigate whether this difference in

55

Chapter Two

molecular weight can be attributed to differences in post-translational modifications, crude membrane fractions from rabbit kidney and Sf9-Mrp2 cells were treated with or without PNGase F and analyzed by immunoblotting using antiserum k78mrp2 (Fig. 18). Deglycosylation reduced the molecular weight of rabbit kidney Mrp2 from ~190 to 175 kDa, which is the size that can be deduced from the rabbit mrp2 cDNA sequence [37,38]. Treatment with PNGase F reduces the molecular mass of Mrp2 in Sf9 cells only slightly to 175 kDa. This indicates that in Sf9 cells, Mrp2 is less glycosylated than in rabbit kidney.

Fig. 2. Comparison of Mrp2 protein levels in Sf9 cells, liver and renal proximal tubules. Protein-equivalents of crude membrane fractions isolated from Sf9-Mrp2 cells (1, 4 ug), rabbit liver (5, 20 ug) and rabbit renal proximal tubules (5, 20 ug) were separated on a 7.5% SDS polyacrylamide gel and subjected to immunoblot analysis using antiserum k78mrp2. Proteins were visualized using enhanced chemiluminescence.

To determine the expression level of rabbit Mrp2 in Sf9 cells, we subjected crude membrane fractions from Sf9-Mrp2 cells, rabbit liver and rabbit renal proximal

Sf9-Mrp2

1 4

5 20

liver

renalproximaltubules


tubular cells to immunoblot analysis using antiserum k78mrp2 (Fig. 2). A 1 jg protein-equivalent of crude membranes from Sf9-Mrp2 cells was sufficient to detect Mrp2. Approximately 20 jg protein-equivalent of crude membranes from liver and renal proximal tubules was needed to detect a similar amount of Mrp2 protein as present in 4 jg protein-equivalent of Sf9-Mrp2 crude membranes.

Fig. 3. Time course of net ATP-dependent uptake of [3H]LTC4 and [3H]E217$G in Sf9-Mrp2 and Sf9- c membrane vesicles. Membrane vesicles from Sf9-c (open symbols) and Sf9-Mrp2 (closed symbols) were incubated in TS-buffer (10 mM Tris-HCl, 250 mM sucrose pH 7.4) at 37 °C for the times indicated. Uptake was determined for [3H]LTC4 (left) and [3H]E217fiG (right) at final concentrations of 3 nM and 50 nM, respectively. Net ATP-dependent transport was calculated by subtracting values in the presence of 4 mM 5'-AMP from those in the presence of 4 mM ATP. Data points represent the mean ± standard error of three or four determinations in a typical experiment.

To investigate whether recombinant rabbit Mrp2 is functional, we investigated uptake of [3H]LTC4 and [3H]E217£G into Sf9-Mrp2 and Sf9-c membrane vesicles. Sf9-Mrp2 membrane vesicles exhibit net ATP-dependent uptake of both [3H]LTC4 (Fig. 3, left) and [3H]E217£G (Fig. 3, right) which was at the 2-min time point ~11- fold higher than in Sf9-c membrane vesicles. In the presence of 5'-AMP, transport of either substrate was hardly detectable in Sf9-Mrp2 membrane vesicles and was

57

Chapter Two

similar to that in Sf9-c membrane vesicles in the presence of 5'AMP or ATP (not shown). Initial rates of uptake for 3 nM [3H]LTC4 and 50 nM [3H]E217£G were 75 and 450 fmol/mg/min, respectively.

1/[SUCROSE] (1/M) [ATP] (mM)

Fig. 4. Osmolarity dependence and effect of ATP concentration on [3H]E217fiG uptake in Sf9-Mrp2 membrane vesicles. Membrane vesicles from Sf9-Mrp2 cells were incubated with 50 nM [3H]E2l7fiG at 37 °C for 1 min. A, ATP-dependent transport was determined in the presence of sucrose concentrations ranging from 250 mM (isotonic condition) to 1000 mM. Initial rates of ATP- dependent [3H]E2l7fiG uptake were plotted against the inverse sucrose concentration in the reaction mixture. B, ATP-dependent transport was determined at various concentrations of ATP (608000 juM). The graph was plotted by fitting the obtained data according to the Michaelis-Menten equation using Prism (GraphPAD, San Diego, CA). Data points in all cases represent the mean ± standard error of three determinations in a typical experiment.

To confirm that vesicle-associated increase of ligand reflects transport into a vesicular space rather than aspecific binding to the membrane, the medium osmolarity dependence of [3H]E217£G uptake was investigated. By increasing the extravesicular sucrose concentration from 250 mM (isotonic condition) to 1000 mM, membrane vesicle space will shrink resulting in decreased uptake. As shown in Fig 4A, initial rates of [3H]E217£G uptake in Sf9-Mrp2 membrane vesicles

58


decreased linearly with increasing concentrations of sucrose. Transport in Sf9-Mrp2 membrane vesicles should also be dependent on the extravesicular concentration of ATP. Fig 4B shows that initial rates of [3H]E217SG uptake increased with ATP concentrations according to Michaelis-Menten kinetics, yielding an apparent Km value of 623 ± 131 uM.

To characterize the inhibitor specificity of rabbit Mrp2, we studied the effect of various compounds on [3H]E217SG uptake by Sf9-Mrp2 membrane vesicles (Table 1). Phenolphthalein glucuronide exerted a profound inhibition, whereas the

other two glucuronides (a-naphthyl-S-D-glucuronide, p-nitrophenyl-S-D- glucuronide) and FL, a substrate of the classic organic anion transport system [34], did not inhibit transport up to 1 mM. Uptake was also susceptible to inhibition by LTC4, MTX and FL-MTX. Furthermore, we tested the LTD4-receptor antagonist MK571 [13] and the immunosuppressive agent CsA, both of which are inhibitors of human MRP1 and rat Mrp2 [3,19] and proved to be inhibitors of rabbit Mrp2.

Discussion

Mrp2 mediates ATP-dependent elimination of conjugated organic anions from liver and has recently been cloned from rat [3,11,22,27] and human [28,36]. We cloned a rabbit cDNA encoding an ABC-transporter that on basis of similar tissue distribution and highest amino acid homology was considered as the rabbit homolog of rat Mrp2 [37,38]. On injection of its cRNAs in Xenopus laevis oocytes, we observed in a few cases a cAMP-dependent chloride conductance [38]. The recent finding that substrates of Mrp2 activate a chloride conductance in hepatocytes of normal rats but not in TR" hepatocytes [40], indicates that this phenomenon warrants further investigation.To investigate Mrp2-mediated transport, we here expressed rabbit Mrp2 in Sf9 cells using recombinant baculovirus. In these cells, Mrp2 is highly expressed, although less glycosylated when compared to kidney Mrp2. This is in line with

59

Chapter Two

TABLE 1Effect of various compounds on Mrp2-mediated [3H]E217ßG transport

Compound Concentration [3H]E217ßGuptake

j M % controlNone (control) 100LTC4 10 22 ± 7ba-Naphthyl-ß-D-glucuronide 1000 94 ± 1p-Nitrophenyl-ß-D-glucuronide 1000 97 ± 5Phenolphthalein glucuronide 1000 5 ± 4bMK571 5 33 ± 3aCyclosporin A (CsA) 10 48 ± 1aFluorescein (FL) 1000 94 ± 4Methotrexate (MTX) 1000 55 ± 4aFluorescein-methotrexate (FL-MTX) 100 22 ± 7b

Sf9-Mrp2 membrane vesicles were incubated with 50 nM [3H]E2l7fiG at 37 °C for 1 min with or without (control) the compounds as indicated. Net ATP-dependent transport was calculated by subtracting values in the presence of 4 mM 5'-AMP from those in the presence of 4 mM ATP. Transport was expressed as percent of the control uptake. Data represent mean values of three to six determinations (± standard error). a p < 0.05; b p < 0.01 (analysis of variance with Bonferroni's correction).

results from other studies in which CFTR and MRP1 were expressed in insect cells and detected as an underglycosylated product [8,15]. Results of functional studies on MRP1- and CFTR-expressing insect cells are comparable to those obtained from transfected eukaryotic cells, indicating that underglycosylation has no significant effect on its function [8,15]. This was further corroborated by inhibition of glycosylation with tunicamycin in drug-resistant MRP1-expressing human cells [1] and, on basis of our studies, can also be concluded for Mrp2.

Based on studies with intact rats and liver canalicular membranes, the conjugates E217SG and LTC4 are considered to be substrates for rat Mrp2 [3,35]. In addition, ATP-dependent transport of LTC4 has been demonstrated in membrane vesicles isolated from NIH/3T3 cells transfected with a rat mrp2 cDNA, and LTC4


efflux was found in Mrp2-expressing Xenopus oocytes and COS-7 cells [12,22]. In the current study, we unambiguously demonstrated that rabbit Mrp2 mediates ATP- dependent uptake of both [3H]E217ßG and [3H]LTC4. The initial uptake rates for [3H]LTC4 and [3H]E217ßG as well as the Vmax value for ATP using [3H]E217ßG as a cosubstrate, are lower than the values described for rat canalicular membrane vesicles [3,39] and membrane vesicles from mrp2-transfected NIH/3T3 cells [12]. This difference, however, may be explained by the substantially lower substrate concentrations that we used. Uptake of [3H]E217ßG in Sf9-Mrp2 membrane vesicles was inhibited by LTC4 and phenolphthalein glucuronide. a-Naphthyl-ß-D- glucuronide and p-nitrophenyl-ß-D-glucuronide had no significant effect on uptake, although both compounds are thought to be Mrp2 substrates. ATP-dependent uptake of p-nitrophenyl-ß-D-glucuronide into rat canalicular membrane vesicles has been described, whereas in TR" rat livers, a-naphthyl-ß-D-glucuronide excretion was

impaired [4,17]. These findings suggest that a-naphthyl-ß-D-glucuronide and p-nitrophenyl-ß-D-glucuronide are transported with low affinity by Mrp2 and consequently, are poor competitive inhibitors themselves. MK571 and CsA are inhibitors of human MRP1 and rat Mrp2 [3,19] and, as shown in this study, also inhibit rabbit Mrp2-mediated [3H]E217ßG transport. However, it remains to be elucidated whether these compounds are Mrp2 substrates.

Mrp2 is expressed not only in liver canalicular membranes [3,27], but also in the brush-border membrane of renal proximal tubular cells [33] and small intestinal villi (see chapter 3). However, the functional identification of an ATP- dependent organic anion transporter in membrane vesicles from renal proximal tubular cells, such as in liver canalicular membranes, has never been documented [29]. This is mainly due to technical limitations because membrane vesicles of renal proximal tubular cells are exclusively orientated right-side out [9]. Recently, Masereeuw et al. [24] identified an energy-dependent transport mechanism for organic anions in isolated renal proximal tubules from killifish using FL-MTX as a substrate. The excretory pathway of FL-MTX was characteristic for its sensitivity to LTC4, MTX, CsA and probenecid. In addition, the energy-dependency of this

61

Chapter Two

pathway was confirmed by treating cells with KCN, which did not influence FL- MTX uptake but completely abolished luminal excretion. This suggests that FL- MTX may be an Mrp2-substrate for which we provide evidence in this study because FL-MTX strongly inhibits [3H]E217SG uptake in Sf9-Mrp2 membrane vesicles. In contrast, FL did not inhibit [3H]E217SG uptake, whereas MTX was only partially inhibitory. It remains to be established whether Mrp2 directly mediates ATP-dependent uptake of FL-MTX.

Besides Mrp2, additional organic anion transporters might be present in brush-border membranes of renal proximal tubular cells. For example, excretion of FL-MTX was shown to be only partially inhibited by probenecid, whereas the probenecid-insensitive mechanism was inhibited completely by verapamil [24]. In addition, it has been shown that TR- rats have impaired hepatic excretion of the

conjugates a-naphthyl-S-D-glucuronide and LTC4, whereas urinary excretion is hardly affected [4,10], suggesting that the deficiency of Mrp2 in the kidney can be compensated for by other organic anion transporters. Possible candidates might be the organic anion transporters Oatpl and Oat-k1, which are both localized in brush-border membranes of renal proximal tubular cells [2,25]. Although Oatpl and Oat-k1 are structurally not related to Mrp2, these proteins mediate transport of Mrp2-substrates, such as E2I 7 SG, LTC4 and MTX [14,20,31]. Furthermore, the recently identified family members of human MRP1 (i.e. MRP3, MRP4, and MRP5) are all expressed to some extent in the kidney and might also be involved in renal organic anion transport [18].

In conclusion, we demonstrated ATP-dependent transport by recombinant rabbit Mrp2 and provided new data on inhibitor specificity. In future studies, this expression system will be used for identification and characterization of Mrp2- substrates, with emphasis on compounds that are excreted by the kidney.

62


Acknowledgments

We thank A. Hartog for isolation of cells of rabbit renal proximal tubules. We also thank Drs. J. Renes and M. Müller (Division of Gastroenterology and Hepatology, University Hospital Groningen, The Netherlands) for stimulating discussions and suggestions for improving the vesicular transport assay.

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25. Masuda, S., H. Saito, H. Nonoguchi, K. Tomita, and K.-I. Inui. (1997). mRNA distribution and membrane localization of the OAT-K1 organic anion transporter in rat renal tubules. FEBS Lett. 407: 127-131

26. Müller, M. and P.L.M. Jansen. (1997). Molecular aspects of hepatobiliary transport. Am. J. Physiol. 272: G1285-G1303.

27. Paulusma, C.C., P.J. Bosma, G.J.R. Zaman, C.T.M. Bakker, M. Otter, G.L. Scheffer, R.J. Scheper, P. Borst, and R.P.J. Oude Elferink. (1996). Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science 271: 1126-1128.

28. Paulusma, C.C., M. Kool, P.J. Bosma, G.L. Scheffer, F. ter Borg, R.J. Scheper, G.N.J. Tytgat, P. Borst, F. Baas, and R.P.J. Oude Elferink. (1997). A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology 25: 1539-1542.

29. Pritchard, J.B. and D.S. Miller. (1993). Mechanisms mediating renal secretion of organic anions and cations. Physiol. Rev. 73: 765-796.

30. Rose, U.M., R.J.M. Bindels, A. Vis, J.W.C.M. Jansen, and C.H. van Os. (1993). The effect of L-type Ca2 + channel blockers on anoxia-induced increases in intracellular Ca 2+ concentration in rabbit proximal tubule cells in primary culture. Pflügers arch. 423: 378-386.

31. Saito, H., S. Masuda, and K.-I. Inui. (1996). Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney. J. Biol. Chem. 271: 20719-20725

32. Sanger, F., S. Nicklen, and A.R. Coulson. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.

33. Schaub, T.P., J. Kartenbeck, J. König, O. Vogel, R. Witzgall, W . Kriz, and D. Keppler. (1997). Expression of the conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubules. J. Am. Soc. Nephrol. 8: 1213-1221.

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34. Sullivan, L.P., J.A. Grantham, L. Rome, D. Wallace, and J.J. Grantham. (1990). Fluorescein transport in isolated proximal tubules in vitro: epifluorometric analysis. Am. J. Physiol. 258: F46-51.

35. Takikawa, H., R. Yamazaki, N. Sano, and M. Yamanaka. (1996). Biliary excretion of estradiol-17 betaglucuronide in the rat. Hepatology 23: 607-613.

36. Taniguchi, K., M. Wada, K. Kohno, T. Nakamura, T. Kawabe, M. Kawakami, K. Kagotani, K. Okumura, S.-I. Akiyama, and M. Kuwano. (1996). A human canalicular multispecific organic anion transporter (cMOAT) gene is overexpressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation. Cancer Res. 56: 4124-4129.

37. van Kuijck, M.A., M. Kool, G.F.M. Merkx, A. Geurts van Kessel, R.J.M. Bindels, P.M.T. Deen, and C.H. van Os. (1997). Assignment of the canalicular multispecific organic anion transporter (cMOAT) gene to human chromosome 10q24 and mouse chromosome 19D2 by fluorescent in situ hybdridisation. Cytogenet. Cell Genet. 77: 285-287.

38. van Kuijck, M.A., R.A.M.H. van Aubel, A.E. Busch, F. Lang, F.G.M. Russel, R.J.M. Bindels, C.H. van Os, and P.M.T. Deen. (1996). Molecular cloning and expression of a cyclic AMP-activated chloride conductance regulator: A novel ATP-binding cassette transporter. Proc. Natl. Acad. Sci. USA 93: 5401-5406.

39. Vore, M., T. Hoffman, and M. Gosland. (1996). ATP-dependent transport of b-estradiol 1 7-(b-D-glucuronide) in rat canalicular membrane vesicles. Am. J. Physiol. 271: G791-G798.

40. Weinman, S.A. and M.W. Carruth. (1997). MRP-2/cMOAT mediates leukotriene-dependent chloride channel activation in hepatocytes. Hepatology 26: 131A (abstract).

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Immunolocalization of the Multidrug Resistance Protein Mrp2 in Rabbit Small Intestine, Liver, and Kidney

Rémon A.M.H. van Aubel1, Anita Hartog2, René J.M. Bindels2,Carel H. van Os2, and Frans G.M. Russel1

Depts. of ’Pharmacology and Toxicology, and 2Cell Physiology, University of Nijmegen, The Netherlands

Adapted version submitted for publication

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Immunolocalization of Rabbit Mrp2

Summary

Multidrug resistance protein 2 (Mrp2) is an ATP-dependent transporter of anionic conjugates. Human MRP2 and rat mrp2 mRNA is abundantly expressed in liver and to a lesser extent in small intestine and kidney. In contrast, highest expression of rabbit mrp2 mRNA is found in small intestine. In this study, we investigated expression and immunolocalization of Mrp2 in rabbit small intestine, liver and kidney. Recombinant rabbit Mrp2 expressed in Sf9 cells was detected using immunocytochemistry and immunoblotting. Crude membranes of various rabbit tissues revealed expression of a 190 kDa protein representing glycosylated Mrp2 only in small intestine, kidney, and liver. In sections of rabbit small intestine, Mrp2-specific immunofluorescence was found at the brush-border (apical) membrane of villi decreasing in intensity from the tip to the villus. Like rat Mrp2 and human MRP2, rabbit Mrp2 is localized to the hepatocyte canalicular (apical) membrane and the brush-border membrane of renal proximal tubules. These results are in line with the postulated function of Mrp2 in drug excretion into the intestinal lumen, urine and bile. In the small intestine, Mrp2 might also function as an absorption barrier limiting the oral bioavailability of drugs.

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Introduction

Multidrug resistance protein 2 (MRP2) is an ATP-dependent drug transporter and member of the ATP-binding cassette (ABC) superfamily [12]. The substrate specificity of rat Mrp2 includes a variety of compounds including unconjugated (bromosulphophthalein, folates) and conjugated (leukotriene C4, LTC4; estradiol- 175-D-glucuronide, E2I 7 SG; bilirubin-glucuronides) organic anions [12,18,40]. These substrates have been identified by comparative studies with wildtype rats and the hyperbilirubinemic rat strains EHBR and GY/TR" defective for functional Mrp2 [2,10,27]. In addition, substrate specificity has been characterized using various types of cell lines overexpressing recombinant rat Mrp2 or human MRP2 [4,6,11,42]. Such transfected cell lines have also indicated that human MRP2 is able to confer resistance to various cationic anti-cancer drugs, such as cisplatin, vincristine, etoposide and methotrexate [4,9,14].

We have cloned a rabbit ABC-transporter, which shows highest identity to MRP2 and functions as an ATP-dependent anionic conjugate transporter upon expression in Sf9 cells [42,43]. In agreement with human and rat, expression of rabbit mrp2 mRNA is found in kidney, liver, and all parts of the small intestine [2, 10,15,43]. However, rabbit mrp2 mRNA is most abundantly expressed in small intestine, whereas highest expression of human MRP2 and rat mrp2 mRNA is found in liver [10,15,43]. Furthermore, immunohistochemistry revealed expression of human and rat MRP2/Mrp2 at the apical membrane of renal proximal tubules [34,35], whereas expression of rabbit Mrp2 was found at the basolateral membrane of renal distal tubules [43]. Therefore, we reanalyzed the expression and localization of Mrp2 in rabbit small intestine, kidney and liver by using immunoblotting and immunohistochemistry.

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Antibodies. Affinity-purified rabbit polyclonal anti-actin antibodies (20-33) were purchased from Sigma Immunochemicals (St. Louis MO, USA). 101E12 is a monoclonal antibody (mAb) directed against the apical membrane of rabbit renal proximal tubules [31]. Polyclonal antibody GP8 was obtained by immunizing guinea pigs with a glutathione 5-transferase (GST) fusion protein containing the 159 carboxyl-terminal amino acids (1405-1564) of rabbit Mrp2 as described [42].

Membrane isolations. All membrane preparations were performed at 4 °C. Rabbit tissues were excised and homogenized in TS-buffer (250 mM sucrose, 10 mM Tris-Hepes, pH7.4). After centrifugation at 200 x g, the supernatant was centrifuged at 100,000 x g for 30 min and the pellet of crude membranes was dissolved in TS-buffer. Sf9 cells were infected with a recombinant baculovirus encoding Mrp2 or the 5-subunit of the rat H+/K+ ATPase (mock) as described [42], washed in phosphate-buffered saline, and resuspended in hypotonic buffer (0.5 mM sodium phosphate, 0.1 mM EDTA, pH 7.0) supplemented with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 5 jg/ml aprotinin, 5 jg/ml leupeptin, 1 mM pepstatin). Cells were stirred gently on ice for 90 min and the resulting lysate was centrifuged at 100,000 x g for 60 min. The pellet of crude membranes was resuspended in TS-buffer using a potter homogenizer and the homogenate was centrifuged at 12,000 x g for 10 min. The postnuclear supernatant was centrifuged at100,000 x g for 40 min and the pellet obtained was resuspended in TS-buffer with a tight- fitting Dounce (type B) homogenizer. The suspension was layered over 38% sucrose in 5 mM Hepes/KOH pH 7.4 and centrifuged at 100,000 x g for 2 h. The interphase was collected and homogenized with a tight-fitting Dounce (type B) homogenizer and the suspension was centrifuged at 100,000 x g for 40 min. The resulting pellet was resuspended in TS-buffer and passed through a 27-gauge needle for 30 times. Membrane vesicles were frozen and stored at -80 °C until use. Protein concentrations were determined using the BioRad protein assay (BioRad Laboratories, Veendendaal, The Netherlands). Membranes were treated with PNGase F (New England Biolabs, Leiden, The Netherlands) according to the manufacturer.

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Immunoblotting. Protein-equivalents (5 jjg) of membrane preparations were solubilized in Laemmli sample buffer, heated at 65 °C for 10 min, separated on a 6% (for detection of Mrp2) or 10% (for detection of fi-actin) polyacrylamide gel, and transferred to Hybond-C pure nitrocellulose membrane (Amersham, Buckinghamshire, UK). Transfer of proteins was confirmed by the reversible staining of the membrane with Ponceau Red. Subsequently, the blot was blocked for 60 min with 5% nonfat dry milk powder in Tris- buffered saline supplemented with 0.3% Tween-20 (TBS-T) and washed twice with TBS-T. The membrane was incubated at 4 0C for 16 h with polyclonal antibodies GP8 or 20-33 diluted 1:1000 in TBS-T. After two times washing for 5 min with TBS-T, the blot was blocked for 30 min as described above. The blot was then washed twice with TBS-T and incubated at room temperature for 60 min with affinity-purified horseradish peroxidase (HRP)-conjugated goat anti-guinea pig IgG or affinity-purified HRP-conjugated goat antirabbit IgG (Sigma Immunochemicals, St. Louis MO) diluted 1:5000 in TBS-T. Finally, the blot was washed twice for 5 min with TBS-T and TBS, respectively. Proteins were visualized using enhanced chemiluminescence (Pierce, Rockford IL).

Immunohistochemistry. Sf9 cells were cultured on coverslips and infected as described

[42]. Cells were fixed in 100% methanol for 10 min at -20 °C and air-dryed. Slices of rabbit small intestine, liver, and kidney were fixed in 1% (v/v) periodate-lysine- paraformaldehyde fixative for 2 h, washed with 20% (w/v) sucrose in phosphate-buffered saline, and subsequently frozen in liquid N2 . Coverslips with Sf9 cells and tissue sections (7 ywM) were blocked with 10% (v/v) goat serum in TBS for 15 min, washed three times with TBS, and incubated with GP8 (diluted 1:50 in TBS) for 16 hours at 4 °C. Kidney sections were also incubated with mAb 101E12 (hybridoma culture medium). After washing in TBS, coverslips and tissue sections sections were incubated with 1:50 diluted tetramethyl rhodamine isothiocyanate (TRITC)-conjugated affinity-purified goat anti-mouse Fab (Biomakor, Rehovot, Israel) and/or fluorescein isothiocyanate (FITC)-conjugated affinity-purified goat anti-guinea pig IgG (Sigma Immunochemicals, St. Louis MO, USA), dehydrated by 100% methanol, mounted in mowiol (Hoechst), and analyzed by immunofluorescence microscopy.

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m

A

*1200 kDa g

116 kDa

Mrp2 mock

Fig. 1. Sf9 cells overexpressing rabbit Mrp2. Sf9 cells were infected with a recombinant baculovirus encoding Mrp2 or the S-subunit of the rat H+/K+ ATPase (mock). A, membrane vesicles from infected Sf9 cells were subjected to immunoblot analysis using polyclonal antibody GP8. Arrow heads indicate molecular weight in kilodalton (kDa). B, Sf9 cells were cultured on coverslips and infected with Mrp2 or H+/K+ ATPase S-subunit (mock) encoding baculovirus. Cells were fixed and incubated with GP8 and analyzed by immunofluorescence microscopy. White marker bar at the bottom right equilizes in length to 38 /jm.

Results

To investigate the immunolocalization of rabbit Mrp2 in small intestine, kidney, and liver, we initially used the rabbit polyclonal antibodies k51mrp2 and k78mrp2, which recognize recombinant rabbit Mrp2 expressed in Sf9 cells as reported previously [42]. However, these antibodies gave rise to high background staining on sections of rabbit tissues. Therefore, a new polyclonal antibody (GP8) was raised in guinea pigs directed against the carboxy-terminus of rabbit Mrp2. In immunoblot analysis, GP8 detected a single band with a molecular weight of ~180 kDa in membrane vesicles from Sf9 cells overexpressing Mrp2 (Fig. 1A). As assessed by treating membranes with PNGase F, the 180 kDa band represents underglycosylated Mrp2 [42]. Furthermore, immunocytochemistry revealed clear

Chapter Three

fluorescence in Sf9 cells overexpressing Mrp2 but not in mock-infected Sf9 cells (Fig. 1B).

Rabbit mrp2 mRNA is expressed in small intestine, kidney and liver [43]. To investigate the tissue distribution of rabbit Mrp2, crude membranes of various rabbit tissues were subjected to immunoblot analysis using GP8. Of all the tissues examined, a protein of approximately 190 kDa was detected in crude membranes from only liver, kidney and small intestine (Fig. 2A). As an internal control, polyclonal antibodies against 5-actin (20-33) detected a protein of about 43 kDa in crude membranes of all tissues examined. PNGase F treatment of crude membranes from liver, kidney, and small intestine, indicated that the immunoreactive band of ~190 kDa represents glycosylated rabbit Mrp2 (Fig. 2B).

The immunolocalization of rabbit Mrp2 was investigated by indirect immunofluorescence using polyclonal antibody GP8. PLP-fixed sections of rabbit duodenum showed intense immunopositive fluorescence at the brush-border membrane of villi (Fig. 3A and B ). The intensity of fluorescence decreased along the length of the villus towards the crypts, at which no staining was observed (Fig 3B and D). Other parts of the small intestine, i.e. submucosa and muscle layers were negative (Fig. 3B and C). Sections incubated with preimmune serum or with GP8 preincubated with the fusion protein antigen revealed no staining (not shown). To confirm that rabbit Mrp2 is the ortholog of human MRP2 and rat Mrp2, we investigated the localization of Mrp2 in rabbit kidney and liver. In PLP-fixed sections of rabbit kidney, Mrp2 expression was confined to the apical membrane of tubules located in the cortex and outer medulla (Fig. 4A). Other parts of the nephron, i.e. glomeruli, cells of Henle's ascending and descending loop, distal cells, and cells of the cortical and medullary collecting duct, were negative.

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E C H Li Lu K S

AMrp2

ß-actin

K Li+ PNGase F

B* • « • 1 190 kDa

175 kDa

Fig 2. Tissue distribution of rabbit Mrp2. A, crude membranes were prepared from rabbit erythrocytes (E), colon (C), heart (H), liver (Li), lung (Lu), kidney (K), and small intestine (S) and subjected to immunoblot analysis using polyclonal antibodies GP8 and 20-33 for detection of rabbit Mrp2 (upper panel) and ß-actin (lower panel), respectively. B, crude membranes of kidney, liver, and small intestine were treated with PNGase F and subjected to immunoblot analysis using polyclonal antibody GP8.

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Fig. 3. Immunolocalization of Mrp2 in rabbit small intestine. PLP-fixed sections of rabbit small intestine were incubated with polyclonal antibody GP8. A and B, Mrp2-specific immunofluorescence was located to the apical membrane of the whole villus. D, high magnification of a crypt area showing decreasing Mrp2-specific immunofluorescence from the lower part of the villus (arrow heads) towards the crypt (arrows). C and E, Phase-contrast microscopy of the section identical to B and D, respectively. White marker bar at the bottom left equalizes in length to 216 jjm in A and B, and 54 jjM in D.

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Fig. 4. Immunolocalization of Mrp2 in rabbit kidney. PLP-fixed sections of rabbit kidney were incubated with polyclonal antibody GP8 preincubated without (A) or with the fusion protein antigen (B). A, low magnification showed Mrp2-specific fluorescence in the apical membrane of S1-S3 proximal tubules. C and D, high magnification of sections double-labeled with GP8 and mAb 101E12. Immunofluorescence specific for Mrp2 and the brush-border membrane are shown in C and D, respectively. White marker bar at the bottom left equalizes in length to 56 /jm.

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Fig. 5. Immunolocalization of Mrp2 in rabbit liver. PLP-fixed sections of rabbit liver were labeled with polyclonal antibody GP8 preincubated without (A) or with the fusion protein antigen (B) and analyzed by immunofluorescence microscopy. Clear Mrp2-specific fluorescence was observed in the hepatocyte canalicular membrane (A ). White marker bar at the bottom right equalizes in length to 56 jum.

Sections incubated with GP8 preincubated with the fusion protein antigen (Fig. 4B) or preimmune serum (not shown) revealed no staining. To confirm the localization of Mrp2, sections were simultaneously incubated with mAb 101E12, which recognizes an epitope in the proximal tubule brush-border membrane of segments S1-S3. Immunofluorescence specific for Mrp2 (Fig. 4C) colocalized with fluorescence specific for the brush-border membrane (Fig. 4D). This colocalization was further corroborated by superimposition of both fluorescences (not shown). In sections of rabbit liver, immunofluorescence specific for Mrp2 revealed a staining characteristic for the canalicular membrane (Fig. 5A). A similar staining has been shown in rat and human liver using a monoclonal antibody directed against rat Mrp2 [27,28]. Polyclonal antibody GP8 pre-incubated with the fusion protein antigen (Fig. 5B) or preimmune serum (not shown) revealed no staining.

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Discussion

We investigated the immunolocalization of rabbit Mrp2. In agreement with the tissue distribution of rabbit mrp2 mRNA, immunoblot analysis showed expression of Mrp2 exclusively in liver, kidney, and small intestine crude membranes. Rabbit Mrp2 was localized to the brush-border membrane of small intestinal villi, and similar to human MRP2 and rat Mrp2, to the hepatocyte canalicular membrane and the brush-border membrane of renal proximal tubules.

Immunoblot analysis of Mrp2 expression in rabbit tissues as shown here is in agreement with that reported for Mrp2 in rat tissues [35]. However, the expression level of rabbit Mrp2 is much higher in kidney than liver, whereas the opposite has been shown for rat Mrp2 [35]. A possible explanation for this difference might be that total crude membranes were used for detection of Mrp2 in rabbit liver, whereas rat Mrp2 was determined in purified canalicular (apical) membrane vesicles [35]. Since the liver canalicular membrane accounts for only 13% of the total plasma membrane, the amount of Mrp2 detected in rabbit liver might be underestimated. Another explanation might be that our antibody cross reacts with other members of the MRP-family. Indeed, human MRP3, MRP4, MRP5, and MRP6 mRNA is expressed in kidney to some extent [13,15,16]. However, MRP3-5 are among other tissues also expressed in lung and colon, but on immunoblot no protein was detected in crude membranes of these tissues using polyclonal antibody GP8. In addition, no proteins were detected in erythrocytes, in which MRP1 and at least another ATP-dependent anionic conjugate transporter is expressed [30,33,44]. Furthermore and of most importance, murine Mrp1 [29], human MRP3 [17], human MRP5 [3], and rat Mrp6 [20] are expressed in basallateral membranes of epithelial cells.

Comparitive studies with wildtype and Mrp2-deficient (GY/TR-, EHBR) rats using isolated canalicular membrane vesicles or perfused livers, have indicated that Mrp2 is important in the biliary excretion of multiple conjugated and unconjugated organic anions [37]. Excretion across the canalicular membrane of

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for example, a-naphthyl-S-D-glucuronide [5], E3040-glucuronide [38,39], cefpiramide [24], and HSR-903 [25] is markedly decreased in livers from EHBR and GY/TR- rats. On the other hand, uptake studies with canalicular membrane vesicles using E3040-glucuronide or pravastatin as a substrate provided clear evidence for an additional ATP-dependent transport system alongside MRP2 [26,45]. Furthermore, biliary excretion of paracetamol glucuronide, which is sensititive to probenecid [32], is not mediated by Mrp2 [41]. The mechanism governing the renal and intestinal excretion of conjugated organic anions by MRP2 is still not elucidated. An ATP-dependent efflux mechanism for the organic anions fluorescein-methotrexate (FL-MTX) and lucifer yellow (LY) has been characterized in killifish renal proximal tubules [21,22]. An Mrp2 ortholog is probably involved, since efflux was partly inhibited by LTC4 and immunohistochemistry using a polyclonal antibody directed against rabbit Mrp2 revealed staining of the killifish proximal tubule brush-border membrane [21-23]. The few data available indicate that, in contrast to their impaired biliary excretion, the renal excretion of

a-naphthyl-S-D-glucuronide [5], E3040-glucuronide [39], cefpiramide [24], and HSR-903 [25] is unaltered in EHBR and GY/TR" rats. This indicates that other transport mechanisms exist for the renal excretion of organic anions, which compensate for the absence of Mrp2. Like urinary excretion, intestinal excretion of

a-naphthyl-S-D-glucuronide in GY/TR- rats is not impaired also suggesting additional transport pathways [5].

Besides Mrp2, the brush-border membrane of renal proximal tubules and small intestine also contains the MDR1-gene encoded drug-efflux pump P-glycoprotein (Pgp) [1,7]. Humans express one gene product, whereas mice have two, i.e. encoded by the mdr1a and mdr1b genes [7]. Pgp transports a variety of compounds in an ATP-dependent fashion, including chemotherapeutic agents, hormones and HIV-1 protease inhibitors [8,19]. Mice with a disruption of the mdr1a gene [mdrla (-/-)], which is normally expressed in small intestine, show a 2-4 fold increase in plasma concentration of indinavir, nelfinavir or saquinavir compared to wildtype mice [19]. Since Mrp2 has been proposed to mediate

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transport of at least the latter compound [8], Mrp2 might also be a determinant in the low oral bioavailability of certain (antiviral) drugs.

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28. Paulusma, C.C., M. Kool, P.J. Bosma, G.L. Scheffer, F. ter Borg, R.J. Scheper, G.N.J. Tytgat, P. Borst, F. Baas, and R.P.J. Oude Elferink. (1997). A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology 25: 1539-1542.

29. Peng, K.-C., F. Cluzeaud, M. Bens, J.-P. Duong van Huyen, M.A. Wioland, R. Lacave, and A. Vandewalle.(1999). Tissue and cell distribution of the multidrug resistance-associated protein (MRP) on mouse intestine and kidney. J. Histochem. Cytochem. 47: 757-767.

30. Pulaski, L., G. Jedlitschky, I. Leier, U. Buchholz, and D. Keppler. (1996). Identification of the multidrug- resistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes. Eur. J. Biochem. 241:644-648.

31. Rose, U.M., R.J.M. Bindels, A. Vis, J.W.C.M. Jansen, and C.H. van Os. (1993). The effect of L-type Ca2 + channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture. Pflügers arch. 423: 378-386.

32. Savina, P.M. and K.L. Brouwer. (1992). Probenecid-impaired biliary excretion of acetaminophen glucuronide and sulfate in the rat. Drug. Metab. Dispos. 20: 496-501.

33. Saxena, M. and G.B. Henderson. (1996). MOAT4, a novel multispecific organic-anion transporter for glucuronides and mercapturates in mouse L1210 cells and human erythrocytes. Biochem. J. 320: 273-281.

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34. Schaub, T.P., J. Kartenbeck, J. König, H. Spring, J. Dorsam, G. Staehler, S. Storkel, W.F. Thon, and D. Keppler (1999). Expression of the MRP2 gene-encoded conjugate export pump in human kidney proximal tubules and in renal cell carcinoma. J. Am. Soc. Nephrol. 10: 1159-1169.

35. Schaub, T.P., J. Kartenbeck, J. König, O. Vogel, R. Witzgall, W. Kriz , and D. Keppler. (1997). Expression of the conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubules. J. Am. Soc. Nephrol. 8: 1213-1221.

36. Schinkel, A.H., U. Mayer, E. Wagenaar, C.A. Mol, L. van-Deemter, J.J. Smit, d. van, V, A.C. Voordouw, H. Spits, O. van-Tellingen, Zijlmans, W.E. Fibbe, and P. Borst. (1997). Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc. Natl. Acad. Sci. USA 94: 4028-4033.

37. Suzuki, H. and Y. Sugiyama. (1998). Excretion of GSSG and glutathione conjugates mediated by MRP1 and cMOAT/MRP2. Semin Liver Dis 18: 359-376.

38. Takenaka, O., T. Horie, K. Kobayashi, H. Suzuki, and Y. Sugiyama. (1995). Kinetic analysis of hepatobiliary transport for conjugated metabolites in the perfused liver of mutant rats (EHBR) with hereditary conjugated hyperbilirubinemia. Pharm. Res. 12: 1746-1755.

39. Takenaka, O., T. Horie, H. Suzuki, and Y. Sugiyama. (1995). Different biliary excretion systems for glucuronide and sulfate of a model compound; study using Eisai hyperbilirubinemic rats. J. Pharmacol. Exp. Ther. 274: 1362-1369.

40. Takikawa, H., R. Yamazaki, N. Sano, and M. Yamanaka. (1996). Biliary excretion of estradiol-17 betaglucuronide in the rat. Hepatology 23: 607-613.

41. van Aubel, R.A.M.H., J.B. Koenderink, J.G.P. Peters, P.M.T. Deen, C.H. van Os, and F.G.M. Russel. (1998). Involvement of the multidrug resistance-associated protein 2 (Mrp2) in the renal excretion of drug glucuronide conjugates (abstract). J. Am. Soc. Nephrol. 9: 305.

42. van Aubel, R.A.M.H., M.A. van Kuijck, J.B. Koenderink, P.M.T. Deen, C.H. van Os, and F.G.M. Russel.(1998). Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance- associated protein Mrp2 expressed in insect cells. Mol. Pharmacol. 53: 1062-1067.

43. van Kuijck, M.A., R.A.M.H. van Aubel, A.E. Busch, F. Lang, F.G.M. Russel, R.J.M. Bindels, C.H. van Os, and P.M.T. Deen. (1996). Molecular cloning and expression of a cyclic AMP-activated chloride conductance regulator: A novel ATP-binding cassette transporter. Proc. Natl. Acad. Sci. USA 93: 5401-5406.

44. Wijnholds, J., R. Evers, M.R. Leusden, C.A. Mol, G.J.R. Zaman, U. Mayer, J. Beijnen, P. van der Valk, P. Krimpenfort, and P. Borst. (1997). Increased sensitivity to anticancer drugs and decreased inflammatory responce in mice lacking the multidrug resistance-associated protein. Nature Med. 3: 1275-1279.

45. Yamazaki, M., K. Kobayashi, and Y. Sugiyama. (1996). Primary active transport of pravastatin across the liver canalicular membrane in normal and mutant Eisai hyperbilirubinaemic rats. Biopharm. Drug Dispos. 17:645-659.

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Mechanisms and Interaction of Vinblastine and Reduced Glutathione Transport in Membrane Vesicles by the Rabbit Multidrug Resistance Protein Mrp2 Expressed in Insect Cells

Remon A.M.H. van Aubel1, Jan B. Koenderink2, Janny G.P. Peters1,Carel H. van Os3, and Frans G.M. Russel1

Depts. o f1Pharmacology and Toxicology, 2Biochemistry, and 3Cell Physiology, University of Nijmegen, The Netherlands

Molecular Pharmacology, 1999, volume 56, pages 714-719

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Vinblastine and GSH Transport by Rabbit Mrp2

Summary

The present study examined how the multidrug resistance protein 2 (MRP2), which is an ATP-dependent anionic conjugate transporter, also mediates transport of the chemotherapeutic cationic drug vinblastine (VBL). W e show that ATP-dependent [3H]VBL (0.2 jM ) uptake into membrane vesicles from Sf9 cells infected with a baculovirus encoding rabbit Mrp2 (Sf9-Mrp2) was similar to vesicles from mock- infected Sf9 cells (Sf9-mock), but could be stimulated by reduced glutathione (GSH) with a half maximum stimulation of 1.9 ± 0.1 mM. At 5 mM GSH, initial ATP-dependent [3H]VBL uptake rates were saturable with an apparent Km of 1.5 ± 0.3 jM . The inhibitory effect of VBL on Mrp2-mediated ATP-dependent transport of the anionic conjugate [3H]leukotriene C4 ([3H]LTC4) was potentiated by increasing GSH concentrations. Membrane vesicles from Sf9-Mrp2 cells exhibited a — 7-fold increase in initial GSH uptake rates compared to membrane vesicles from Sf9-mock cells. Uptake of [3H]GSH was osmotically sensitive, independent of ATP, and trans-inhibited by GSH. The anionic conjugates estradiol-17S-D- glucuronide and LTC4 cis-inhibited [3H]GSH uptake but only in the presence of ATP. Whereas ATP-dependent [3H]VBL uptake was stimulated by GSH, VBL did not affect [3H]GSH uptake. In conclusion, our results show that GSH is required for Mrp2-mediated ATP-dependent VBL transport and that Mrp2 transports GSH independent of VBL.

(available online at http://molpharm.aspetjournals.org)

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Introduction

The multidrug resistance protein 2 (MRP2; or canalicular multispecific organic anion transporter, cMOAT) is an ATP-dependent anionic conjugate transporter, which is expressed in small intestine and apical (canalicular) membranes of hepatocytes and renal proximal tubules (for review see ref. 13). Substrates of MRP2 include glutathione S-conjugates, such as S-(dinitrophenyl)-glutathione (DNP-SG), S-(prostaglandin Ai)-glutathione and leukotriene C4 (LTC4) and glucuronide conjugates of bilirubin and estradiol [7,10,11,21,32]. MRP2 belongs to a branch of at least six multidrug resistance proteins (MRP1-MRP6) within the superfamily of ATP-binding cassette (ABC) proteins (see for latest update: http://www.med.rug.nl/mdl/tab3.htm). Involvement in the phenotype of multidrug resistance, however, has only been proven for MRP1 because it is frequently overexpressed in various drug-selected cancer cell lines and transfection of drug- sensitive cells with an MRP1 cDNA results in resistance to various drugs (for review see ref. 6). Recently, overexpression of MRP2 mRNA and MRP2 protein levels has been found in a few cancer cell lines selected for cis- diamminedichloroplatin (CDDP) [15]. Downregulation of endogenous MRP2 protein levels in HepG2 cells via antisense transfection reduces resistance to various chemotherapeutic drugs, including vincristine (VCR) and CDDP [14]. MDCKII cells stably transfected with an MRP2 cDNA show enhanced vinblastine (VBL) transport across the apical membrane [7].

The mechanism by which MRP2 mediates transport of cationic chemotherapeutic drugs is unknown. Since MRP1 requires reduced glutathione (GSH) for ATP-dependent VCR uptake into membrane vesicles [19], MRP2- mediated drug transport might also be dependent on GSH. Furthermore, it has been suggested that GSH itself is an MRP2 substrate, but reports are contradictory [8,24]. The rat strains EHBR and TR", which lack functional Mrp2, are characterized by hyperbilirubinemia as well as low biliary GSH contents compared to wildtype rats [13,24,30]. However, GSH uptake into EHBR liver

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canalicular membrane vesicles has been reported to be similar to wildtype liver canalicular membrane vesicles [8]. Furthermore, both wildtype and EHBR canalicular membrane vesicles only harbor ATP-independent GSH transport systems, suggesting that Mrp2 is not involved in GSH transport [2,8].

The purpose of this study was to investigate the mechanism by which MRP2 mediates ATP-dependent transport of the cationic drug VBL. By using membrane vesicles from Sf9 cells overexpressing rabbit Mrp2 as described previously [32], we determined uptake of [3H]VBL with emphasis on a putative role for GSH. In addition, we investigated whether GSH itself is an Mrp2 substrate and how VBL and GSH affect Mrp2-mediated ATP-dependent uptake of [3H]LTC4.


Materials. [G-3H]VBL sulphate (15.5 Ci/mmol) was purchased from Amersham International (Buckinghamshire, UK). [14, 15, 19, 20-3H]LTC4 (165 Ci/mmol) and [glycine-2-3H]GSH (44.8 Ci/mmol) were purchased from NEN Life Science Products (Hoofddorp, The Netherlands). E217GG, LTC4 , glucuronate, BSA, S-methyl-GSH, GSH, ATP, DTT and VBL were purchased from Sigma (Zwijndrecht, The Netherlands). Acivicin was purchased from ICN Biochemicals (Cleveland, Ohio, USA). Creatine phosphate and creatine kinase were purchased from Boehringer Mannheim (Almere, The Netherlands). Glassfiber GF/F filters were purchased from Whatmann (Omnilabo International, Breda, The Netherlands). ME-25 membrane filters were purchased from Schleicher and Schuell (Dassel, Germany). MK571 was a generous gift of Dr. A.W. Ford-Hutchinson (Merck Frosst, Center for Therapeutic Research, Quebec, Canada).

Expression of Mrp2 in Sf9 cells and isolation of membrane vesicles. Sf9 cells (106/ml) were grown as 100 ml suspension cultures and infected for three days at a multiplicity of infection of 1-5 with a recombinant baculovirus encoding rabbit Mrp2 (Sf9-Mrp2) as described recently [32]. For control experiments, Sf9 cells were infected with a baculovirus encoding the G-subunit of rat H+/K+ ATPase (Sf9-mock). Membrane vesicles were isolated as described [32]. Briefly, cells were collected, resuspended in hypotonic

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buffer (0.5 mM sodium phosphate, 0.1 mM EDTA, pH 7.0) and centrifuged at 100,000 x g for 30 min. The pellet of crude membranes was resuspended in TS-buffer (10 mM Tris- Hepes, 250 mM sucrose pH 7.4) and centrifuged at 12,000 x g for 10 min. The postnuclear supernatant was centrifuged at 100,000 x g for 40 min and the pellet obtained was resuspended in TS-buffer, layered over 38% sucrose in 5 mM Hepes/KOH pH 7.4 and centrifuged at 100,000 x g for 120 min. The interphase was collected, homogenized and centrifuged at 100,000 x g for 40 min. The resulting pellet was resuspended in TS- buffer and passed through a 27-gauge needle for 30 times. Membrane vesicles were frozen and stored at -80 °C until use.

Transport studies. Uptake of [3H]VBL into membrane vesicles was performed as described recently [9,32], with modifications. Briefly, membrane vesicles (20 jg protein-equivalent) were thawed for 1 min at 37 °C and added to pre-warmed TS-buffer supplemented with an ATP-regeneration mix (4 mM ATP, 20 mM MgCb, 10 mM creatine phosphate, 100 jg/ml creatine kinase) and 0.2 jM [3H]VBL in a final volume of 60 j l . The reaction mixture was incubated at 37 °C and at indicated times samples were taken from the mixture, diluted in 1 ml ice-cold TS-buffer and filtered through Whatman GF/F filters (soaked overnight in 5% (w/v) bovine serum albumin at 37 °C) using a filtration device (Millipore Corp., Bedford, MA). Filters were washed once with ice-cold 5 ml TS-buffer and dissolved in liquid scintillation fluid to determine the bound radioactivity. Uptake of [3H]LTC4 at 2 nM was performed as described [32]. Uptake of [3H]GSH at 37 °C was performed according to the same procedure as for [3H]LTC4, except that 5 mM DTT was added at every GSH concentration used and that samples were filtered through ME-25 membrane filters. Uptake of GSH was not affected by 250 jM acivicin, indicating

negligible activity of y-glutamyltransferase in membrane vesicles of Sf9 cells. In all uptake experiments, net ATP-dependent transport was calculated by subtracting values in the absence of ATP from those in the presence of ATP. Measurements were corrected for the amount of ligand bound to the filters (usually < 2% of total radioactivity).

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TABLE 1ATP-dependent uptake of [3H]VBL into membrane vesicles of Sf9-Mrp2 and Sf9-mock cells

Compound ATP-dependent [3H]VBL uptake

Sf9-Mrp2 Sf9-mockpmol/mg/2 min pmol/mg/2 min

Control 5.0 ± 1.0 5.1 ± 1.2GSH 23.1 ± 0̂

0. 4.9 ± 0.5GSH + MK571 12.0 ± 1 1 a,b n.d.5-methyl-GSH 15.2 ± 0. 4.6 ± 0.7DTT 4.8 ± 1.2 5.3 ± 0.9Glucuronate 4.9 ± 0.8 5.0 ± 1.2

Membrane vesicles were incubated in TS-buffer with 0.2 j M [3H]VBL and an ATP- regenerating system at 37 °C for 2 min in the absence or presence of the compounds as listed below. All compounds were used at a concentration of 5 mM, except for MK571 (0.1 mM). ATP-dependent [3H]VBL uptake was calculated by subtracting values in the absence of ATP from those in the presence of 4 mM ATP. Data are mean ± S.E. from two experiments performed in triplicate. ap < 0.01 compared to control; bp < 0.01 compared to uptake in the presence of GSH but absence of MK571 (analysis of variance with Bonferroni's correction); n.d., not determined.

Results

We investigated uptake of [3H]VBL into membrane vesicles prepared from Sf9 cells infected with a baculovirus containing a rabbit mrp2 cDNA (Sf9-Mrp2). We have recently shown that these membrane vesicles contain functional Mrp2 as assessed by uptake studies with [3H]LTC4 and estradiol-17£-D-[3H]glucuronide [32]. As shown in Table 1, ATP-dependent uptake of 0.2 j M [3H]VBL into Sf9-Mrp2 membrane vesicles was 5 pmol/mg/2 min and did not differ from uptake into membrane vesicles from Sf9 cells infected with a baculovirus encoding the £- subunit of rat H+/K+ ATPase (Sf9-mock). In the presence of 5 mM GSH, however, ATP-dependent uptake of [3H]VBL into Sf9-Mrp2 membrane vesicles increased

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TIME (min) TIME (min)

Fig. 1. Time course of GSH-stimulated ATP-dependent [3H]VBL transport by Mrp2. Membrane vesicles prepared from Sf9-Mrp2 and Sf9-mock cells were incubated in TS-buffer with 0.2 j M

[3H]VBL and 5 mM GSH at 37 °C. Samples were taken from the reaction mixture for the times indicated. A, GSH-stimulated [3H]VBL uptake into membrane vesicles from Sf9-Mrp2 in the absence (open circles) or presence (closed circles) of ATP. B, net GSH/ATP-dependent [3H]VBL uptake into membrane vesicles of Sf9-mock (open squares) and Sf9-Mrp2 (closed squares) cells. Data are mean ± S.E. from two experiments performed in triplicate.

è l i 25-

QzLUCLmQICLI—<ÌCO0

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(N"raE"oE3LU

£CL3

20-

1 2 3 4

1/[SUCROSE] (1/M)

0

Fig. 2. Osmolarity dependency of GSH- stimulated ATP-dependent [3H]VBL transport by Mrp2. GSH/ATP-dependent [3H]VBL uptake into Sf9-Mrp2 membrane vesicles was determined for 2 min in the presence of sucrose concentrations ranging from 250 mM (isotonic condition) to 1000 mM. Net GSH/ATP-dependent [3H]VBL transport was plotted against the inverse sucrose concentration in the reaction mixture. Linear fitting of the obtained data was performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego CA, USA). Data are mean ± S.D. from one experiment performed in triplicate.

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5-fold. The leukotriene D4-receptor antagonist MK571 [12], which is an inhibitor of Mrp2-mediated transport [5,32], inhibited GSH-stimulated ATP-dependent [3H]VBL uptake by ~ 60 %. S-methyl-GSH also increased ATP-dependent [3H]VBL uptake, however, this stimulation was 55% as compared with GSH. Glucuronate and dithiothreitol (DTT), which was used to maintain GSH in the reduced form [2], did not stimulate uptake.

In the presence of GSH and ATP, Sf9-Mrp2 membrane vesicles exhibited time- dependent uptake of [3H]VBL (Fig. 1A). In the absence of ATP, GSH-stimulated [3H]VBL uptake into Sf9-Mrp2 membrane vesicles was low. For Sf9-Mrp2 membrane vesicles, initial uptake rates of net GSH-stimulated ATP-dependent [3H]VBL were 5.2 + 0.7 pmol/mg/20 sec (Fig. 1ß). In contrast, transport into Sf9- mock membrane vesicles was independent of time (4.9 + 0.5 pmol/mg) and probably reflects unspecific membrane binding. To confirm that vesicle-associated increase of ligand reflects transport into a vesicular space, the effect of medium osmolarity on [3H]VBL uptake was investigated (Fig. 2). GSH-stimulated ATP- dependent [3H]VBL uptake into Sf9-Mrp2 membrane vesicles decreased linearly with increasing concentrations of sucrose. Since vesicle space decreases with increasing osmolarity, extrapolation of the data to zero vesicle space indicates that 5.0 + 0.6 pmol VBL/mg protein is bound to the vesicle membrane.

To further investigate GSH-stimulated VBL transport by Mrp2, initial transport rates were determined at various concentrations of [3H]VBL (Fig. 3A). In the presence of 5 mM GSH, initial ATP-dependent [3H]VBL uptake rates increased with increasing VBL concentrations. Fitting of the obtained data according to the Michaelis-Menten equation revealed a Km of 1.5 + 0.3 jM and Vmax of 89 + 6 pmol/mg/20 sec. We also investigated the GSH concentration dependency of Mrp2-mediated ATP-dependent VBL transport (Fig 3ß). Initial ATP-dependent [3H]VBL uptake rates increased with increasing GSH concentrations to a maximum at 10 mM. According to Michaelis-Menten kinetics, half-maximum stimulation was determined at a GSH concentration of 1.9 + 0.1 mM.

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[GSH] (mM)

Fig. 3. VBL and GSH concentration dependency of GSH-stimulated ATP-dependent [3H]VBL transport by Mrp2. A, membrane vesicles prepared from Sf9-Mrp2 cells were incubated at 37 °C for 20 sec in TS-buffer supplemented with 5 mM GSH and [3H]VBL concentrations ranging from 0.2- 5 /jM. B, Sf9-Mrp2 membrane vesicles were incubated at 37 °C for 20 sec in TS-buffer supplemented with 0.2 j M [3H]VBL and GSH concentrations ranging from 0.5 - 40 mM. All data were corrected for binding of VBL to the vesicle membrane and were fitted according to the Michaelis-Menten equation using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego CA, USA). Data are mean from two experiments performed in triplicate (± S.E.).

We investigated the effect of GSH and VBL on ATP-dependent uptake of the high- affinity substrate LTC4 into Sf9-Mrp2 membrane vesicles. As shown in Fig. 4, initial rates of ATP-dependent [3H]LTC4 uptake were not inhibited but rather stimulated by GSH at concentrations of 0.1, 1 and 5 mM. VBL (0.1 mM) was only a poor inhibitor of initial [3H]LTC4 uptake rates. However, the inhibitory effect of VBL (20%) was greatly stimulated in the presence of 0.1, 1 and 5 mM GSH to 72, 82 and 95%, respectively.

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VBL ^ + “ + - + - +

no 0.1 mM 1 mM 5 mM GSH GSH GSH GSH

Fig 4. Effect of GSH and VBL on Mrp2- mediated [3H]LTC4 transport. Sf9-Mrp2 membrane vesicles were incubated with 2 nM [3H]LTC4 at 23 °C for 30 sec in the absence (control) or presence of VBL (0.1 mM) and/or GSH at concentrations as indicated. Transport was expressed as percent inhibition of net ATP-dependent [3H]LTC4 uptake in the absence of GSH and VBL. Final concentrations of the dissolvent dimethylsulfoxide were < 0.1%. Data are mean ± S.E. from two experiments performed in triplicate.

To investigate whether GSH itself is an Mrp2 substrate, we measured uptake of [3H]GSH at concentrations of 0.1 and 5 mM. Sf9-Mrp2 membrane vesicles exhibited time-dependent uptake of [3H]GSH at 0.1 mM, however, uptake was only modestly increased as compared to uptake into Sf9-mock membrane vesicles (Fig. 5A). At 5 mM, [3H]GSH was taken up into Sf9-Mrp2 membrane vesicles with an initial rate of 1.4 ± 0.3 nmol/mg/min, which was ~ 7-fold higher compared to initial rates in Sf9-mock membrane vesicles (Fig. 58). In the presence of ATP, time- dependent uptake of [3H]GSH (0.1 or 5 mM) into Sf9-Mrp2 and Sf9-mock membrane vesicles was not significantly different from uptake in the absence of ATP (Fig. 5^ and 8). Uptake of [3H]GSH was sensitive to osmolarity, indicating that vesicle-associated [3H]GSH levels represent true uptake into membrane vesicles rather than binding to the vesicle membrane (Fig. 5C).

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TIME (min)

1/[SUCROSE] (1/M)

Fig. 5. Time course and osmolarity dependency of Mrp2-mediated [3H]GSH transport. A and B, membrane vesicles prepared from Sf9-Mrp2 (squares) and Sf9- mock (circles) cells were incubated at 37 °C in TS-buffer with [3H]GSH at concentrations of 0.1 mM (A) or 5 mM (B) in the presence (closed symbols) or absence (open symbols) of ATP. Data are mean ± S.E. from two experiments performed in triplicate.C, [3H]GSH uptake into Sf9-Mrp2 membrane vesicles was determined for 4 min in the presence of sucrose concentrations ranging from 250 mM (isotonic condition) to 1000 mM. [3H]GSH transport was plotted against the inverse sucrose concentration in the reaction mixture. Linear fitting of the obtained data was performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego CA, USA). Data are mean ± S.D. from one experiment performed in triplicate.

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Preloading of Sf9-Mrp2 membrane vesicles with 5 mM GSH reduced initial [3H]GSH uptake rates by ~85% either in the presence or absence of ATP (Table 2). Initial [3H]GSH uptake rates were also inhibited by LTC4, estradiol-17S-D- glucuronate (E2I 7SG) and MK571 but only in the presence of ATP, whereas S-methyl-GSH had no effect (Table 2).

TABLE 2Effect of various compounds on [3H]GSH uptake into membrane vesicles of Sf9-Mrp2 cellsCompound [3H]GSH uptake

+ ATP -ATPControl 100 100GSH (5 mM, trans) 12 ± 3a 15 ± 4aS-methyl-GSH (5 mM) 98 ± 5 1 0 0 + 2LTC4 (5 ^M) 23 ± 5a 9 7 + 1 7E2 I 7 SG (0.1 mM) 50 + 12a 8 9 + 1 2MK571 (0.1 mM) 32 + 7a 9 9 + 7VBL (0.2 j jM) 9 8 + 5 9 8 + 1 0VBL (0.1 mM) 1 0 2 + 3 1 0 7 + 3

Membrane vesicles were incubated in TS-buffer with 5 mM [3H]GSH at 37 °C for 1 min in the absence or presence of ATP supplemented with or without (control) the compounds as listed below. The trans effect of GSH was studied by preloading Sf9-Mrp2 membrane vesicles with 5 mM GSH for 1 hour at 37 °C and resuspending them in TS-buffer with [3H]GSH. Transport was expressed as percent of the control. Data are mean ± S.E. from two experiments performed in triplicate. ap < 0.01 (analysis of variance with Bonferroni's correction). Control uptake of [3H]GSH into Sf9-Mrp2 membrane vesicles was 1.3 ± 0.2 nmol/mg/min in the presence or absence of ATP.

Recently, Loe et al. have demonstrated that VCR stimulates GSH uptake into membrane vesicles from MRP7-transfected HeLa cells, providing evidence for a VCR/GSH cotransport mechanism [20]. In view of a possible VBL/GSH cotransport mechanism by Mrp2, we investigated the effect of various VBL concentrations on uptake of [3H]GSH into Sf9-Mrp2 membrane vesicles. However, the initial uptake rate of [3H]GSH at 0.1 mM (95 ± 12 pmol/mg/min; n = 2) or 5 mM (1.3 ± 0.2

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nmol/mg/min; n = 2) was not affected by either 0.2 jM , 10 jM or 0.1 mM VBL (Table 2 and not shown). Furthermore, none of these VBL concentrations affected uptake of [3H]GSH at 5 mM for 4 min (not shown).

Discussion

The present study demonstrates that the ATP-dependent anionic conjugate transporter Mrp2 requires GSH for ATP-dependent transport of the cationic drug VBL. S-Methyl-GSH also stimulated ATP-dependent VBL transport but to a lesser extent than GSH, whereas, glucuronate was not able to induce VBL transport. These results are in line with previous reports showing that MRP1-mediated VCR transport is stimulated by GSH and to a lesser extent by S-methyl-GSH but not by glucuronate [19,20]. Furthermore, we show that stimulation of Mrp2-mediated ATP-dependent VBL transport by GSH is saturable with a half maximum stimulation at 1.9 mM. In presence of 5 mM GSH, saturability of transport is observed at increased VBL concentrations with a Km of 1.5 jM . Moreover, the small inhibitory effect of VBL on Mrp2-mediated ATP-dependent [3H]LTC4 transport was potentiated by increasing the GSH concentration.

Secondly, we show that membrane vesicles from Sf9-Mrp2 cells exhibited a — 7-fold increase in initial GSH uptake rates compared to membrane vesicles from Sf9-mock cells. Uptake of GSH was sensitive to medium osmolarity, independent of ATP and almost completely abolished when membrane vesicles were preloaded with GSH. Initial GSH uptake rates in Sf9-Mrp2 membrane vesicles were inhibited by the anionic conjugates LTC4 and E217SG but only in the presence of ATP. These results suggest that Mrp2 is permeable for GSH and that the site for GSH permeability interacts with the site for active transport of anionic conjugates. In this respect, MK571 inhibits both Mrp2-mediated ATP-dependent anionic conjugate transport and ATP-independent GSH transport (ref. 32 and this study). Similarly to our results, GSH permeability has recently been shown for the ATP- dependent chloride channel CFTR, which belongs to the MRP branch of ABC-

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transporters [18]. Alternatively, Mrp2 itself may not be involved in GSH transport but may regulate an endogenous ATP-independent GSH transporter. Such a regulatory mechanism for Mrp2 has been proposed based on results from uptake studies with liver canalicular membrane vesicles [2,8,22]. Radiation inactivation of canalicular GSH transport has indicated the complexity of low-affinity GSH transport, which involves multiple putative transporters including presumably Mrp2 [22]. Furthermore, low-affinity GSH uptake into liver canalicular membrane vesicles is ds-inhibited by DNP-SG in the absence of ATP but trans-stimulated by DNP-SG in the presence of ATP [2,8]. The characteristics of this Mrp2-regulated GSH transport, however, are different from our results, because anionic conjugates inhibited GSH uptake into Sf9-Mrp2 membrane vesicles in the presence of ATP, whereas they did not affect uptake in the absence of ATP.

A recent report by Paulusma et al. is at variance with the hypothesis of Mrp2 as both an ATP-dependent anionic conjugate transporter and an ATP-independent GSH transporter [25]. These authors demonstrated that inhibition of ATP synthesis in MRP2-transfected MDCKII cells greatly reduced efflux of both DNP-SG and GSH across the apical membrane [7,25]. Nonetheless, a difference in uptake of GSH between membrane vesicles of transfected and parental cells either in the presence or absence of ATP was undetectable [25]. Similar contradictory results obtained with intact cells and isolated membrane vesicles have been reported for MRP1. Whereas expression levels of MRP1 correlate with efflux of GSH from intact cells [25,26,33], MRP1-mediated uptake of GSH into membrane vesicles either in the presence or absence of ATP could not be demonstrated [16,20]. It has recently been suggested that MRP1 and MRP2 might mediate GSH efflux from intact cells in the form of a short-lived complex with an intracellular compound, which is lacking in preparations of membrane vesicles [25,33]. Such a mechanism would explain why Mrp2-deficient rats have very low biliary GSH levels, although GSH uptake into EHBR canalicular membrane vesicles is intact [8,24]. On the other hand, membrane vesicle preparations from various cell lines and tissues may be unsuitable to detect GSH uptake because of their mixed orientation and

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Chapter Four

possible leakage. Recently, Rebbeor et al. have demonstrated GSH uptake into yeast sec6-4 membrane vesicles [27], which are orientated almost exclusively inside-out [1]. Uptake of GSH into these vesicles was saturable (Km = 15-20 mM), competitively inhibited by DNP-SG (Ki = —0.2 mM), and dependent on ATP. A further study identified the yeast cadmium factor 1 (YCF1), a yeast ortholog of mammalian MRP1 and MRP2 [17], as the major contributor to ATP-dependent GSH transport [28]. However, membrane vesicles from wildtype yeast also show low levels of ATP-independent GSH uptake and at present it is unknown whether YCF1 or a different transporter is involved [27,28].

The physiological significance of MRP2-mediated GSH secretion into bile and urine is not clear. Biliary and urinary GSH may provide protection against oxidative damage and, in addition, may be a source for glutamate, glycine and cysteine after degradation of GSH by extracellular membrane-bound peptidases [23]. Furthermore, urinary GSH may account for detoxification of agents that enter the urine directly via glomerular filtration. Besides MRP2, also other transport proteins are involved in biliary GSH secretion [3]. In liver canalicular membranes, the putative low and high affinity ATP-independent GSH transporters both have an estimated molecular weight of — 70 kDa [22]. Although their molecular identity is unknown, these transporters might be canalicular isoforms of the 74 kDa sinusoidal organic anion transporting polypeptide (Oatp1), which functions as a GSH/organic solute exchanger [3].

In this study, we tried to address the mechanism by which GSH affects Mrp2- mediated ATP-dependent VBL transport. Possible explanations for the effect of GSH are spontaneous formation of a glutathione-S-conjugate, a cotransport mechanism or an indirect interaction. So far, conjugates of GSH with vinca alkaloids, such as VCR and VBL, have not been demonstrated [4,31]. Furthermore, HPLC analysis of the products excreted by MRP1 -transfected SW-1573/S1 cells after preloading with VCR only revealed unmodified drugs [33]. Thus, either GSH is cotransported with VBL or GSH indirectly stimulates ATP-dependent VBL transport. GSH stimulates Mrp2-mediated ATP-dependent VBL transport with an

100


apparent half maximum stimulation at —2 mM. A similar value (2.7 mM) was found for GSH-stimulated ATP-dependent daunorubicin transport by MRP1 [29]. The transport affinity of GSH for MRP2/Mrp2 appears to be 10-fold lower ( — 20 mM), which is in the same range as reported for YCF1 [25,28]. This suggests that VBL enhances the affinity of GSH for Mrp2, but we could not demonstrate that VBL induces Mrp2-mediated GSH transport as one would expect for a cotransport mechanism. Loe et al. [20] recently have shown both GSH-stimulated VCR and VCR-stimulated GSH uptake into membrane vesicles of HeLa cells overexpressing human MRP1, suggesting a VCR/GSH cotransport mechanism. VBL also stimulated GSH uptake into MRP1 enriched membrane vesicles, although to a lesser degree than VCR [20]. With respect to our results, we cannot exclude that it is impossible to detect a VBL-dependent stimulation of GSH transport above the background of GSH uptake, because of the relatively low affinity and high Vmax of the latter process. However, even if VBL does not stimulate GSH transport, we conclude from our data that Mrp2-mediated GSH transport does not require the cotransport of VBL.

References

1. Ambesi, A., K. E. Allen, and C. W . Slayman. (1997). Isolation of transport-competent secretory vesicles from Saccharomyces cerevisiae. Anal. Biochem. 251: 127-129.

2. Ballatori, N. and W . J. Dutczak. (1994). Identification and characterization of high and low affinity transport systems for reduced glutathione in liver cell canalicular memebranes. J. Biol. Chem. 269: 19731-19737.

3. Ballatori, N. and J. F. Rebbeor (1998). Roles of MRP2 and oatp1 in hepatocellular export of reduced glutathione. Semin Liver Dis. 18: 377-387.

4. Ban, N., Y. Takahashi, T. Takayama, T. Kura, T. Katahira, S. Sakamaki, and Y. Niitsu (1996). Transfection of glutathione S-transferase (GST)-pi antisense complementary DNA increases the sensitivity of a colon cancer cell line to adriamycin, cisplatin, melphalan, and etoposide. Cancer Res. 56: 3577-3582.

5. Büchler, M., J. König, M. Brom, J. Kartenbeck, H. Spring, T. Horie, and D. Keppler (1996). cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J. Biol. Chem. 271: 15091-15098.

6. Cole, S. P. C. and R. G. Deeley (1998). Multidrug resistance mediated by the ATP-binding cassette transport protein MRP. Bioessays 20: 931-940.

7. Evers, R., M. Kool, L. van Deemter, H. Janssen, J. Calafat, L. C. J. M. Oomen, C. C. Paulusma, R. P. J. Oude Elferink, F. Baas, A. H. Schinkel, and P. Borst. (1998). Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney M DCK cells expressing cM O AT (MRP2) cDNA. J. Clin. Invest. 101: 1310-1319.

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8. Fernandez-Checa, J. C., H. Takikawa, T. Horie, M. Ookhtens, and N. Kaplowitz (1992). Canalicular transport of reduced glutathione in normal and mutant eisai hyperbilirubinemic rats. J. Biol. Chem. 267: 1667-1673.

9. Horio, M., M. M. Gottesman, and I. Pastan (1988). ATP-dependent transport of vinblastine in vesicles from human multidrug- resistant cells. Proc. Natl. Acad. Sci. USA 85: 3580-3584.

10. Ito, K., H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, and Y. Sugiyama (1998). Functional analysis of a canalicular multispecific organic anion transporter cloned from rat liver. J. Biol. Chem. 273: 1684-1688.

11. Jedlitschky, G., I. Leier, U. Buchholz, J. Hummel-Eisenbeiss, B. Burchell, and D. Keppler (1997). ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2. Biochem. J. 327: 305-310.

12. Jones, T. R., R. Zamboni, M. Belley, E. Champion, L. Charette, A. W . Ford Hutchinson, R. Frenette, J. Y. Gauthier, S. Leger, P. Masson, C. S. McFairlane, H. Piechuta, J. Rokach, H. Williams, and R. N. Young (1989). Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist. Can. J. Physiol. Pharmacol. 67: 17-28.

13. Keppler, D. and J. König (1997). Expression and localisation of the conjugate export pump encoded by the MRP2 (cMRP/cMOAT) gene in liver. FASEB J. 11: 509-516.

14. Koike, K., T. Kawabe, T. Tanaka, S. Toh, T. Uchiumi, M. Wada, S.-I. Akiyama, M. Ono, and M. Kuwano (1997). A canalicular multispecific organic anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells. Cancer Res. 57: 5475-5479.

15. Kool, M., M. de Haas, G. L. Scheffer, R. J. Scheper, M. J. T. van Eijk, J. A. Juijn, F. Baas, and P. Borst (1997). Analysis of expression of cM O AT (MRP2), MRP3, MRP4, and MRP5, homologous of the multidrug resistance- associated protein gene (MRP1) in human cancer cell lines. Cancer Res. 57: 3537-3547.

16. Leier, I., G. Jedlitschky, U. Buchholz, M. Center, S. P. C. Cole, R. G. Deeley, and D. Keppler (1996). ATP- dependent glutathione disulphide transport mediated by the M RP gene-encoded conjugate export pump. Biochem. J. 314: 433-437.

17. Li, Z.-S., M. Szczypka, Y.-P. Lu, D. J. Thiele, and P. A. Rea (1996). The yeast cadmium factor protein (YCF1) is vacuolar glutathione S-conjugate pump. J. Biol. Chem. 271: 6509-6517.

18. Linsdell, P. and J. W . Hanrahan (1998). Glutathione permeability of CFTR. Am. J. Physiol. 44: C323-C326.19. Loe, D. W ., K. C. Almquist, R. G. Deeley, and S. P. C. Cole (1996). Multidrug resistance protein (MRP)-mediated

transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. J. Biol. Chem. 271: 9675-9682.20. Loe, D. W ., R. G. Deeley, and S. P. C. Cole (1998). Characterization of vincristine transport by the M(r) 190,000

multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione. Cancer Res. 58: 51305136.

21. Madon, J., U. Eckhardt, T. Gerloff, B. Stieger, and P. J. Meier (1997). Functional expression of the rat liver canalicular isoform of the multidrug resistance-associated protein. FEBS Lett. 406: 75-78.

22. Mittur, A. V., N. Kaplowitz, E. S. Kempner, and M. Ookhtens (1998). Novel properties of hepatic canalicular reduced glutathione transport revealed by radiation inactivation. Am. J. Physiol. 37: G923-G930.

23. Ookhtens, M. and N. Kaplowitz (1998). Role of the liver in interorgan homeostasis of glutathione and cyst(e)ine. Semin Liver Dis 18: 313-329.

24. Oude Elferink, R. P. J., R. Ottenhoff, W . Liefting, J. de Haan, and P. L. M. Jansen (1989). Hepatobiliary transport of glutathione and glutathione conjugate in rats with hereditary hyperbilirubinemia. J. Clin. Invest. 84: 476-483.

25. Paulusma, C. C., M. van Geer, R. Evers, M. Heijn, R. Ottenhoff, P. Borst, and R. P. J. Oude Elferink (1999). Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione. Biochem. J. 338: 393-401.

26. Rappa, G., A. Lorico, R. A. Flavell, and A. C. Sartorelli (1997). Evidence that the multidrug resistance protein (MRP) functions as a co-transporter of glutathione and natural product toxins. Cancer Res. 57: 5232-5237.

27. Rebbeor, J. F., G. C. Connolly, M. E. Dumont, and N. Ballatori (1998). ATP-dependent transport of reduced glutathione in yeast secretory vesicles. Biochem. J. 334: 723-729.

28. Rebbeor, J. F., G. C. Connolly, M. E. Dumont, and N. Ballatori (1998). ATP-dependent transport of reduced glutathione on YCF1, the yeast orthologue of mammalian multidrug resistance associated proteins. J. Biol. Chem. 273: 33449-33454.

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29. Renes, J., E. G. E. de Vries, E. F. Nienhuis, P. L. M. Jansen, and M. Müller (1999). ATP- and glutathione- dependent transport of chemotherapeutic drugs by the multidrug resistance protein M RP1. Br. J. Pharmacol. 126: 681-688.

30. Takikawa, H., N. Sano, T. Narita, Y. Uchida, M. Yamanaka, T. Horie, T. Mikami, and O. Tagaya (1991). Biliary excretion of bile acid conjugates in a hyperbilirubinemic mutant Sprague-Dawley rat. Hepatology 14: 352-360, 1991.

31. Tew, K. D. Glutathione-associated enzymes in anticancer drug resistance. Cancer Res. 54: 4313-4320.32. van Aubel, R. A. M. H., M. A. van Kuijck, J. B. Koenderink, P. M. T. Deen, C. H. van Os, and F. G. M. Russel

(1998). Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance- associated protein Mrp2 expressed in insect cells. Mol. Pharmacol. 53: 1062-1067.

33. Zaman, G. J. R., J. Lankelma, J. Beijnen, O. van Tellingen, H. Dekker, C. C. Paulusma, R. P. J. Oude Elferink, F. Baas, and P. Borst (1995). Role of glutathione in the export of compounds from cells by the multidrug resistance- associated protein. Proc. Natl. Acad. Sci. USA 92: 7690-7694.

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The Multidrug Resistance Protein Mrp2 Mediates ATP-

Dependent Transport of the Classical Renal Organic Anion

p-Aminohippurate

Remon A.M.H. van Aubel1, Janny G.P. Peters1, Rosalinde Masereeuw1, Carel H. van Os2, and Frans G.M. Russel1

Depts. of Pharmacology and Toxicology and 2Cell Physiology,University of Nijmegen, The Netherlands

Submitted for publication

105

ATP-Dependent PAH Transport by Mrp2

Summary

p-Aminohippurate (PAH) is widely used as a model substrate to characterize organic anion transport in kidney proximal tubules. The carrier responsible for uptake of PAH across the basolateral membrane has been cloned and well characterized, whereas transporters mediating PAH excretion across the brush- border (apical) membrane are still elusive. The multidrug resistance protein 2 (Mrp2) is an ATP-dependent organic anion transporter expressed in liver, small intestine, and at the proximal tubule brush-border membrane. In this study, we show that membrane vesicles from Sf9 cells overexpressing rabbit Mrp2 exhibit saturable ATP-dependent [14C]PAH uptake (Km = 1.7 ± 0.4 mM, Vmax = 8 .6 ± 1.5 nmol/mg/5 min). The organic anions leukotriene C4 (K = 1.0 ± 0.4 jM ), estradiol- 175-D-glucuronide, lucifer yellow, FURA-AM, fluorescein-methotrexate, and probenecid inhibit ATP-dependent [14C]PAH uptake. In conclusion, Mrp2 is the first renal apical organic anion transporter proven to mediate PAH transport.

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Chapter Five

Introduction

The kidney plays an important role in the clearance of endogenous compounds and xenobiotics from the body. The proximal tubule is the primary site at the nephron where organic anions are taken up from the blood and excreted into urine. p-Aminohippurate (PAH) is widely used as a model substrate to investigate renal handling of organic anions because of its high renal clearance and low susceptibility to metabolism [19]. Uptake of PAH from the blood across the basolateral membrane is mediated by the PAH/dicarboxylate exchanger OAT1 [4,10,23,26,27]. This is a so-called tertiary active process coupled indirectly via Na+/dicarboxylate cotransport to the Na+ gradient, which is maintained by the Na+/K+ ATPase [18,24]. Studies with isolated membrane vesicles have shown that excretion of PAH across the brush-border (apical) membrane involve potential- driven facilitated transport as well as an anion exchange mechanism [15,20]. Both systems have not yet been identified at the molecular level. The cloned organic anion transporters Oatp1 and Oat-k1 , which have been localized to the brush- border membrane, do not appear to transport PAH [1,6,14,21].

The multidrug resistance protein 2 (MRP2) is an ATP-dependent organic anion transporter located at the apical membrane of hepatocytes, small intestinal villi, and renal proximal tubules (refs. [7,22] and van Aubel et al., unpublished). Substrates of MRP2 include conjugated organic anions, such as leukotriene C4

(LTC4), estradiol-175-D-glucuronide (E217SG), and bilirubin-glucuronide, and unconjugated organic anions, such as bromosulfophthalein and folates [25]. In this study, we investigated whether MRP2 may also mediate ATP-dependent transport of PAH. Therefore, we determined uptake of [14C]PAH into membrane vesicles from Sf9 cells overexpressing recombinant rabbit Mrp2.

108



Materials. [14C]PAH (53.1 mCi/mmol) and [6,7-3H]E2175G (55 Ci/mmol) were purchased from NEN Life Science Products (Hoofddorp, The Netherlands). Creatine phosphate and creatine kinase were purchased from Boehringer Mannheim (Almere, The Netherlands). Glass fiber GF/F filters were purchased from Whatmann (Omnilabo International, Breda, The Netherlands). All other chemicals were obtained from Sigma (Zwijndrecht, The Netherlands).

Sf9-Mrp2 and control cells. Sf9 cells expressing rabbit Mrp2 (Sf9-Mrp2) were generated by infection of cells with a recombinant baculovirus encoding Mrp2 as described previously [28]. Briefly, Sf9 cells (106/ml) were grown as 100 ml suspension cultures and infected for three days at a multiplicity of infection of 1-5 with Mrp2-encoding baculovirus. For control experiments, Sf9 cells infected with a baculovirus encoding the 5-subunit of rat H+/K+ ATPase were used (Sf9-mock).

Transport studies. Membrane vesicles from Sf9-Mrp2 and control cells were isolated as described [28]. Uptake of [14C]PAH (50 jM ) or [3H]E2175G (50 nM) into membrane vesicles was performed as described [28]. Briefly, membrane vesicles (40 jg protein- equivalent) were thawed for 1 min at 37 °C and added to pre-warmed TS-buffer (10 mM Tris-Hepes/ 250 mM sucrose pH 7.4) supplemented with an ATP-regeneration mix (4 mM ATP, 10 mM MgCl2, 10 mM creatine phosphate, 100 jg/ml creatine kinase) and a labeled compound in a final volume of 60 j l . The reaction mixture was incubated at 37 °C and at indicated times samples were taken from the mixture, diluted in 1 ml ice-cold TS-buffer and filtered through Whatman GF/F filters using a filtration device (Millipore Corp., Bedford, MA). Filters were washed once with ice-cold 5 ml TS-buffer and dissolved in liquid scintillation fluid to determine the radioactivity bound. In control experiments, ATP was omitted from the reaction mixtures. Net ATP-dependent transport was calculated by subtracting values in the absence of ATP from those in the presence of ATP. Measurements were corrected for the amount of ligand bound to the filters (usually < 2% of total radioactivity).

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Chapter Five

Determination of kinetic parameters. ATP-dependent [14C]PAH uptake into membrane vesicles of Sf9-Mrp2 cells could be described as uptake via a Michaelis-Menten process in parallel with nonspecific uptake. The Mrp2-mediated component of uptake was defined as the difference between ATP-dependent uptake in membrane vesicles of Sf9-Mrp2 and Sf9- mock cells. Inhibition of LTC4 against ATP-dependent [14C]PAH uptake was analyzed assuming competitive inhibition to a one binding site model. The inhibition constant (Ki) was calculated according to the equation:

Ki = IC50/(1 + (S/Km))where IC50 is the concentration of LTC4 at half maximal inhibition, S is the [14C]PAH concentration, and Km is the Michaelis-Menten constant of Mrp2-mediated ATP-dependent [14C]PAH uptake. Curve fitting was done by least squares nonlinear regression analysis using the GraphPad software (GraphPad Prism version 3.0 for Windows, GraphPad Software, San Diego, USA).

Results

We investigated uptake of [14C]PAH into membrane vesicles prepared from Sf9 cells infected with a baculovirus containing a full-length rabbit mrp2 cDNA (Sf9- Mrp2). W e have recently shown that these membrane vesicles contain functional Mrp2 as assessed by ATP-dependent uptake of the anionic conjugates [3H]LTC4 and [3H]E2175G [28]. The time course of [14C]PAH uptake into these vesicles is shown in Fig. 1. In the presence of ATP, membrane vesicles from Sf9-Mrp2 cells exhibited uptake of [14C]PAH, whereas in the absence of ATP, uptake was negligible (Fig. 1A). The non-metabolizable ATP analogue 5'-AMP did not stimulate uptake of [14C]PAH (not shown). Membrane vesicles of mock-infected Sf9 cells (Sf9-mock) exhibited a relatively high basal uptake of [14C]PAH in the presence of ATP. Net ATP-dependent [14C]PAH uptake in membrane vesicles of Sf9-Mrp2 and Sf9-mock cells was linear with time up to 5 min with initial rates of

110

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4C]P

AH

AT

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PTA

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(p

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(pm

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[14C

]PAH

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PTA

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1/[sucrose] (1/M )

Fig. 1. Time course and osmolarity dependency of Mrp2-mediated [,4C]PAH uptake. Membrane vesicles prepared from Sf9-Mrp2 and Sf9-mock cells were incubated in TS-buffer with 50 jjM [14C]PAH at 37 °C and samples were taken from the reaction mixture for the times indicated.A, uptake of [14C]PAH into Sf9-Mrp2 membrane vesicles in the absence (▲) or presence (■) of ATP.B, net ATP-dependent [14C]PAH uptake into membrane vesicles of Sf9-mock (□) and Sf9- Mrp2 (■) cells. Data are the mean ± S.E. from two experiments performed in triplicate.C, ATP-dependent [14C]PAH uptake into membrane vesicles of Sf9-mock (□) and Sf9- Mrp2 (■) cells was determined for 5 min in the presence of sucrose concentrations ranging from 250 mM (isotonic condition) to 1000 mM. Transport of [14C]PAH was plotted against the inverse sucrose concentration in the reaction mixture. Linear fitting of the obtained data was performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego CA, USA). Data are mean ± S.D. from one experiment performed in triplicate.

111

Chapter Five

116 ± 20 pmol/mg/5 min and 64 ± 10 pmol/mg/5 min, respectively (Fig. 1B). As assessed by uptake studies at increasing extravesicular osmolarities, [14C]PAH values represent uptake into the vesicular space of both Sf9-mock and Sf9-Mrp2 membrane vesicles and little binding (< 2%) was observed (Fig. 1C).

Initial rates of ATP-dependent [14C]PAH uptake in membrane vesicles of Sf9- Mrp2 and Sf9-mock cells increased with increasing PAH concentrations (Fig 2A). Values obtained using Sf9-Mrp2 membrane vesicles were corrected for basal ATP- dependent [14C]PAH uptake as observed in Sf9-mock membrane vesicles and were fitted to the Michaelis-Menten equation. Parameter values (mean ± S.E.) obtained were a Km of 1.7 ± 0.4 mM and a Vmax of 8 .6 ± 1.5 nmol/mg/5 min for Mrp2- mediated ATP-dependent PAH transport (Fig. 2B).

LTC4 is a high-affinity substrate for rat/human Mrp2/MRP2 (Km ~ 1 jM ) [2]. As shown in Fig. 3, LTC4 inhibited Mrp2-mediated ATP-dependent [14C]PAH uptake in a concentration-dependent manner, and curve fitting gave a Ki value of 1.0 ± 0.4 jM . At 3 jM , LTC4 inhibited Mrp2-mediated ATP-dependent [14C]PAH uptake to basal levels observed in membrane vesicles of Sf9-mock cells.

We further investigated the inhibitory effects of various other organic anions on Mrp2-mediated ATP-dependent [14C]PAH uptake (Table 1). For comparison, we tested the same compounds for their ability to inhibit ATP-dependent [3H]E217£G uptake by Mrp2. E217SG, at a two-fold higher concentration than [14C]PAH, was a strong inhibitor of ATP-dependent [14C]PAH uptake (82% inhibition). In contrast, [3H]E217£G uptake rates were inhibited for 35% by PAH at a concentration that exceeded the [3H]E217£G concentration to four orders of magnitude. Probenecid, which is an inhibitor of organic anion transport, inhibited [14C]PAH and [3H]E217£G uptake rates to a similar or lower extent than fluorescein-methotrexate.

112


[PAH] (|JM)

[PAH] (j M)

Fig. 2. Kinetic analysis of Mrp2-mediated ATP-dependent [,4C]PAH uptake. A, membrane vesicles from Sf9-mock (Q and Sf9-Mrp2 (■) cells were incubated with [14C]PAH at 50-400 jM for 5 min. Lines through the data points are the fitted lines according to Michaelis-Menten kinetics in parallel with nonspecific transport. B, corrected Mrp2-specific ATP-dependent [14C]PAH uptake. Data are the mean ± S.E. from two experiments performed in triplicate.

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Chapter Five

Fig. 3. Effect of LTC4 on ATP- dependent [14C]PAH uptake.Membrane vesicles from Sf9-Mrp2 cells were incubated with 50 /j M [14C]PAH for 5 min in the absence or presence of 100, 250, 500, 1000, or 3000 nM LTC4. Data are the mean ± S.E. from two experiments performed in triplicate. Line through the data points is the fitted line according to a one-site competitive model.

0 ' ' 2 3 4

log [LTC4] (nM)

TABLE 1Effect of various organic anions on ATP-dependent uptake of [14C]PAH and [3H]E2176G into Sf9-Mrp2 membrane vesicles

Compound ATP-dependent transport

[14C]PAH [3H]E217gG% control % control

None (control) 100 100E217£G (0.1 mM) 18 ± 8 ndPAH (1 mM) nd 65 ± 9probenecid (0.1 mM) 90 ± 4a 39 ± 9FL-MTX (10 yvM) 51 ± 9 38 ± 8FURA-AM (10 yvM) 75 ± 2 3 ± 1LY (10 yvM) 79 ± 1 5 ± 2

Membrane vesicles from Sf9-Mrp2 cells were incubated with [14C]PAH (50 j M) or [3H]E2l7fiG (50 nM) in the absence (control) or presence of the compounds as indicated. Net ATP-dependent transport was calculated by subtracting values in the absence of ATP from those in the presence of ATP. Transport was expressed as percent of the control uptake. Data are mean (± S.D.) from a typical experiment performed in triplicate. All data were significantly different from controls (p<0.01; p<0.05a, analysis of variance with Bonferroni's correction). nd, not determined.

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150

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50

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114


Discussion

The present study demonstrates that the classical model compound for renal organic anion secretion, PAH, is a substrate for Mrp2, which is expressed at the brush-border membrane of renal proximal tubules. By using membrane vesicles from Sf9 cells expressing rabbit Mrp2, we demonstrated that [14C]PAH uptake is time- and ATP-dependent. In addition, Mrp2-mediated ATP-dependent [14C]PAH uptake was saturable with a Km value of 1.7 ± 0.4 mM. The affinity of PAH for Mrp2 is low in comparison with DNP-SG (0.18 jM ), LTC4 (1 jM ), and E217SG (7 jM ), but higher as compared to reduced glutathione (20 mM) [2,5,16]. Furthermore, LTC4 inhibited Mrp2-mediated PAH transport with a Ki value of 1.0 ± 0.4 jM , which is consistent with a Km of approximately 1 jM determined for rat/human Mrp2/MRP2 [2]. In a preliminary report, it has been shown that membrane vesicles from human MRP7-transfected HeLa cells also exhibit ATP- dependent PAH transport [8]. In contrast to Mrp2, Mrp1 is not expressed in proximal tubules but located at the basolateral membrane of cells of renal distal tubules [17,29].

Little is known about the molecular identity of the pathways for organic anion transport at the brush-border membrane. Studies in brush-border membrane vesicles using PAH as a substrate, have indicated the presence of at least two transport systems, i.e. an anion exchange mechanism and potential-driven facilitated diffusion [15,20]. ATP-dependent PAH uptake has never been demonstrated in these vesicles. This may be explained by the inaccessibility of the ATP-binding site because of the almost complete rightside-out orientation of brush- border membrane vesicles from proximal tubular cells [3]. In intact killifish renal proximal tubules an energy-dependent efflux pathway has been characterized for the organic anions FL-MTX and LY [11,12]. Luminal efflux of FL-MTX and LY was inhibited by the Mrp2 substrates LTC4 and DNP-SG in a concentration-dependent manner [11,12]. Furthermore, immunohistochemistry with an antibody directed against rabbit Mrp2 revealed staining of the brush-border membrane [13]. This

115

Chapter Five

indicates that an ortholog of rabbit Mrp2 is involved in luminal excretion of FL- MTX and LY in killifish proximal tubules. This is further corroborated by our finding that FL-MTX and LY appeared to be good inhibitors of Mrp2-mediated ATP- dependent transport of PAH and E217SG.

The contribution of Mrp2 to overall urinary excretion of PAH is still elusive. Efflux of FL-MTX from killifish proximal tubules is marginally inhibited by PAH [12]. However, this may reflect a difference in affinity, since FL-MTX appeared to be a strong inhibitor of Mrp2-mediated ATP-dependent PAH transport at a concentration 5-fold lower than tracer PAH, suggesting that FL-MTX is a high- affinity substrate unlike PAH. In contrast to FL-MTX, efflux of LY is almost completely inhibited by PAH but only partly by LTC4, which may be explained by involvement of multiple organic anion transporters [11]. Efflux of the mercapturic acid derivative of monochlorobimane (Nac-MCB-Cys) from killifish proximal tubules is also inhibited by LTC4 and PAH [11]. However, it is not yet known whether Nac-MCB-Cys is also a substrate for Mrp2 alongside S-(MCB)-glutathione

[9].Previous studies with isolated renal membrane vesicles have shown the

existence of two ATP-independent low-affinity (Km 5-7 mM) pathways for the efflux of PAH across the brush-border membrane [15,20]. The present results indicate that Mrp2 may be a third low-affinity pathway mediating the ATP-dependent renal excretion of PAH. Whether Mrp2 contributes significantly to urinary PAH excretion will depend on its density at the brush-border membrane as compared to other systems.

Acknowledgments

We thank J.B. Koenderink (Dept. Biochemistry, University of Nijmegen) for expert technical assistance.

116


References

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2. Cui, Y., J. König, U. Buchholz, H. Spring, I. Leier, and D. Keppler. (1999). Drug resistance and ATP-dependentconjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol. Pharmacol. 55: 929-937.

3. Haase, W ., A. Schafer, H. Murer, and R. Kinne. (1978). Studies on the orientation of brush-border membranevesicles. Biochem. J. 172: 57-62.

4. Hosoyamada, M., T. Sekine, Y. Kanai, and H. Endou. (1999). Molecular cloning and functional expression of amultispecific organic anion transporter from human kidney. Am. J. Physiol. 276: F122-F128.

5. Ito, K., H. Suzuki, T. Hirohashi, K. Kume , T. Shimizu, and Y. Sugiyama. (1998). Functional analysis of a canalicular multispecific organic anion transporter cloned from rat liver. J. Biol. Chem. 273: 1684-1688.

6. Kanai, N., R. Lu, Y. Bao, A .W . Wolkoff, and V.L. Schuster. (1996). Transient expression of oatp organic aniontransporter in mammalian cells: identification of candidate substrates. Am. J. Physiol. 270: F319-F325.

7. Keppler, D. and J. König. (1997). Expression and localisation of the conjugate export pump encoded by the MRP2(cMRP/cMOAT) gene in liver. FASEB J. 11: 509-516.

8. Keppler, D., J. König, T.P. Schaub, and I. Leier. Molecular basis of ATP-dependent transport of anionic conjugatesin kidney and liver. In: Renal and hepatic transport - Similarities and Differences, edited by C. Fleck, W . Klinger, and D. Muller. Halle: Nova acta leopoldina, (1998). p. 213-221.

9. Kinoshita, S., H. Suzuki, K. Ito, K. Kume , T. Shimizu, and Y. Sugiyama. (1998). Transfected rat cM O AT is functionally expressed on the apical membrane in Madin-Darby canine kidney (MDCK) cells. Pharm. Res. 15: 1851-1856.

10. Lu, R., B.S. Chan, and V.L. Schuster. (1999). Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C. Am. J. Physiol. 276: F295-F303.

11. Masereeuw, R., M.M. Moons, B.H. Toomey, F.G.M. Russel, and D.S. Miller. (1999). Active Lucifer Yellow secretion in renal proximal tubule: evidence for organic anion transport system crossover. J. Pharmacol. Exp. Ther. 289: 1104-1111.



14. Masuda, S., H. Saito, H. Nonoguchi, K. Tomita, and K.-I. Inui. (1997). mRNA distribution and membrane localization of the OAT-K1 organic anion transporter in rat renal tubules. FEBS Lett. 407: 127-131.

15. Ohoka, K., M. Takano, T. Okano, S. Maeda , K. Inui, and R. Hori. (1993). p-Aminohippurate transport in rat renal brush-border membranes: a potential-sensitive transport system and an anion exchanger. Biol. Pharm. Bull. 16: 395-401.

16. Paulusma, C.C., M. van Geer, R. Evers, M. Heijn, R. Ottenhoff, P. Borst, and R.P.J. Oude Elferink. (1999). Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione. Biochem. J. 338: 393-401.

17. Peng, K.-C., F. Cluzeaud, M. Bens, J.-P. Duong van Huyen, M.A. Wioland, R. Lacave, and A. Vandewalle. (1999). Tissue and cell distribution of the multidrug resistance-associated protein (MRP) on mouse intestine and kidney. J. Histochem. Cytochem. 47: 757-767.

18. Pritchard, J.B. (1988). Coupled transport of p-aminohippurate by rat kidney basolateral membrane vesicles. Am. J. Physiol. 255: F597-F604.

19. Pritchard, J.B. and D.S. Miller. (1993). Mechanisms mediating renal secretion of organic anions and cations. Physiol. Rev. 73: 765-796.

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20. Russel, F.G.M., P.E. van der Linden, W .G . Vermeulen, M. Heijn, C.H. van Os, and C.A. van Ginneken. (1988). Na+ and H + gradient-dependent transport of p-aminohippurate in membrane vesicles from dog kidney cortex. Biochem. Pharmacol. 37: 2639-2649.

21. Saito, H., S. Masuda, and K.-I. Inui. (1996). Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney. J. Biol. Chem. 271: 20719-20725.

22. Schaub, T.P., J. Kartenbeck, J. König, H. Spring, J. Dorsam, G. Staehler, S. Storkel, W .F. Thon, and D. Keppler.(1999). Expression of the MRP2 gene-encoded conjugate export pump in human kidney proximal tubules and in renal cell carcinoma. J. Am. Soc. Nephrol. 10: 1159-1169.

23. Sekine, T., N. Watanabe, M. Hosoyamada, Y. Kanai, and H. Endou. (1997). Expression cloning and characterization of a novel multispecific organic anion transporter. J. Biol. Chem. 272: 18526-18529.

24. Shimada, H., B. Moewes, and G. Burckhardt. (1987). Indirect coupling to Na+ of p-aminohippurate acid uptake into rat renal basolateral membrane vesicles. Am. J. Physiol. 253: F795-F801.

25. Suzuki, H. and Y. Sugiyama. (1998). Excretion of GSSG and glutathione conjugates mediated by MRP1 and cMOAT/MRP2. Semin. Liver. Dis. 18: 359-376.

26. Sweet, D.H., N.A. Wolff, and J.B. Pritchard. (1997). Expression cloning and characterization of ROAT1. The basolateral organic anion transporter in rat kidney. J. Biol. Chem. 272: 30088-30095.

27. Uwai, Y., M. Okuda, K. Takami, Y. Hashimoto, and K.-I. Inui. (1998). Functional characterization of the rat multispecific organic anion transporter OAT1 mediating basolateral uptake of anionic drugs in the kidney. FEBS Lett. 438: 321-324.

28. van Aubel, R.A.M.H., M.A. van Kuijck, J.B. Koenderink, P.M.T. Deen, C.H. van Os, and F.G.M. Russel. (1998). Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance-associated protein Mrp2 expressed in insect cells. Mol. Pharmacol. 53: 1062-1067.

29. Wijnholds, J., G.L. Scheffer, M. vanderValk, P. vanderValk, J.H. Beijnen, R.J. Scheper, and P. Borst. (1998). Multidrug resistance protein 1 protects the oropharyngeal mucosal layer and the testicular tubules against drug- induced damage. J. Exp. Med. 188: 797-808.

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Summary,

General Conclusions and

Future Perspectives

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Summary, General Conclusions & Future Perspectives

Summary

Renal organic anion transport systems play an important role in the elimination of endogenous waste products and xenobiotics, many of which are potentially harmful to the body. In the kidney, the proximal tubule is the site where organic anions are taken up from the blood across the basolateral membrane. This uptake involves an active transport mechanism, because organic anions are transported against a steep electrochemical gradient. Therefore, uptake is the rate-limiting step in excretion of organic anions into the urine. Much attention has been paid to the organic anion uptake process, using p-aminohippurate (PAH) as a model substrate in studies with isolated proximal tubules, basolateral membrane vesicles, or perfused kidney. On the other hand, the transport systems mediating efflux of organic anions across the brush-border membrane have been studied less well. This is in contrast with the field of liver research, in which organic anion transporters involved in biliary efflux of bile acid and non-bile acid organic anions across the canalicular (apical) membrane have been well characterized. In the hepatocyte, excretion of organic anions is directed against a very high concentration gradient maintained in bile and therefore requires active transport systems. Studies with isolated canalicular membrane vesicles, cultured hepatocytes, and perfused liver, have indicated that biliary excretion of non-bile acid organic anions is mediated by an ATP-dependent transport system designated as the canalicular multispecific organic anion transport (cMOAT) system. Without knowing its molecular identity, substrate characteristics of cMOAT have been investigated in comparative transport studies with livers from wildtype rats and two natural mutant rat strains (GY/TR- and EHBR) defective in cMOAT. These mutant rats develop mild conjugated hyperbilirubinemia or congenital jaundice as a consequence of defective cMOAT activity and hepatic accumulation of bilirubinglucuronides.

The cloning of the gene encoding the protein responsible for cMOAT was initiated from a different field of research. Resistance of cancer cells to

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chemotherapeutic drugs is a major problem in treating patients with chemotherapy. The phenotype of multidrug resistance (MDR) is associated with overexpression of the ATP-binding cassette (ABC) family members (MDR1-gene encoded) P-glycoprotein (Pgp) and multidrug resistance protein 1 (MRP1). Overexpression of either Pgp or MRP1 in drug-sensitive cells results in resistance to various chemotherapeutics. The link with cMOAT was made when, virtually at the same time, two groups reported evidence that membrane vesicles from human MRPI-transfected cells exhibit ATP-dependent uptake of anionic conjugates, such as S-(dinitrophenyl)-glutathione and leukotriene C4 (LTC4) (Müller et al., 1994, Proc. Natl. Acad. Sci. 91, 13033; Leier et al., 1994, J. Biol. Chem. 269, 27807). Since biliary excretion of these compounds is markedly decreased in GY/TR" and EHBR rats, but mrpl gene expression in wildtype rat liver is very low, cMoat was proposed as a homolog of rat Mrp1. Indeed, three groups reported the cloning of cMoat as a novel member of the ABC-family with highest identity to MRP1 (Büchler et al., 1996, J. Biol. Chem. 271, 15091; Paulusma et al., 1996, Science 271, 1126; Ito et al., 1997, Am. J. Physiol. 272, G16). In livers of GY/TR- and EHBR, cMoat appeared to be absent from the canalicular membrane and sequence analysis revealed a mutation causing a premature termination of the cmoat gene product. Because of its structural and functional homology to MRP1 and the subsequent cloning of several other human MRP-homologs (MRP3-MRP6), cMOAT was renamed MRP2. Besides in liver, MRP2 is also expressed in the kidney and small intestine. In the kidney, MRP2 is located to the brush-border membrane of renal proximal tubules. Membrane vesicles of the brush-border membrane, however, are exclusively orientated right-side-out with the binding site for ATP directed intravesicularly, which explains why ATP-dependent organic anion transport activity has never been found in this preparation.

In the investigation described in this thesis, we functionally expressed rabbit Mrp2 in Sf9 cells to characterize its transport properties and to use this model system to identify putative substrates, with emphasis on compounds that are excreted by the kidney. In chapter 1, we give a concise overview of the properties

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of organic anion transporters located at the basolateral and brush-border membrane of the renal proximal tubule. Various transport systems have been characterized at the physiological level. Some of these transport systems have also been cloned, such as the PAH/dicarboxylate exchanger Oat1, the anion/sulfate exchanger Sat1, the peptide transporters PEPT1 and 2, and the nucleoside transporters CNT1 and 2. For others, such as Mrp1, Mrp2, Oatpl, Oat-k1, and Oat-k2, a function in the kidney remains to be established. Finally, the homologs Oat2/-3, Oatp3, and MRP3-MRP6 have been cloned but their subcellular localization in the kidney is still elusive.

In chapter 2, we investigated whether rabbit Mrp2 functions as an ATP- dependent organic anion transporter. Therefore, we overexpressed rabbit Mrp2 in Spodoptera frugiperda (Sf9) cells using a baculovirus expression system. In membranes of Sf9 cells, Mrp2 was detected as an underglycosylated protein of ~180 kDa. The expression level of Mrp2 in membranes of Sf9 cells was — 4-fold higher as compared to its expression level in kidney and liver membranes. By using a rapid filtration technique, Mrp2-mediated transport activity was assessed in isolated membrane vesicles supplemented with an ATP-regenerated system. Estradiol-17S-D-[3H]-glucuronide ([3H]E217SG) and [3H]LTC4 were taken up ATP- dependently into membrane vesicles of Sf9-Mrp2 cells but not into control vesicles from mock-infected cells. Evidence was provided for actual uptake of [3H]E217SG into the vesicles rather than binding. Furthermore, Mrp2-mediated [3H]E217SG uptake was saturable for the ATP concentration (Km —0.6 mM). Several compounds (LTC4, MK571, cyclosporin A, methotrexate, fluorescein-methotrexate, phenolphthalein glucuronide) inhibited Mrp2-mediated ATP-dependent

[3H]E217SG uptake, whereas others had no effect (a-naphthyl-S-D-glucuronide, p-nitrophenyl-S-D-glucuronide, fluorescein).

In the initial cloning paper [van Kuijck MA, van Aubel RAMH, Busch AE, Lang F, Russel FGM, Bindels RJM, van Os CH, Deen PMT (1996), Proc. Natl. Acad. Sci. 93, 5401], we reported localization of Mrp2 to the basolateral membrane of rabbit renal distal tubules, although others have shown that in rat kidney, Mrp2

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expression is confined to the apical membrane of the proximal tubule. In chapter 3, we reanalyzed the subcellular localization of rabbit Mrp2 with a novel polyclonal antibody generated in guinea pigs. Mrp2 was detected in Sf9 cells infected with an Mrp2-encoding baculovirus, as well as in kidney, liver and small intestine. Other tissues, such as lung, heart, colon, and erythrocytes, in which various MRP-homologs are expressed, were negative. In rabbit kidney and liver, Mrp2 was located to the apical membrane of proximal tubules and hepatocytes. In addition, Mrp2 was localized at the brush-border membrane of intestinal villi, whereas crypts were negative.

Overexpression of human MRP2 in drug-sensitive cells results in resistance to various chemotherapeutic (cationic) drugs, in a manner similar to human MRP1. Studies with membrane vesicles have indicated that MRP1 mediates ATP- dependent transport of the vinca alkaloid vincristine only in the presence of physiological concentrations ( — 5 mM) of reduced glutathione (GSH). Furthermore, comparative studies with wildtype and GY/TR" livers have suggested that rat Mrp2 transports GSH itself. In chapter 4, we investigated whether rabbit Mrp2 is able to transport the vinca alkaloid vinblastine (VBL) with emphasis on the role of GSH. Membrane vesicles of Sf9-Mrp2 cells exhibited ATP-dependent uptake of [3H]VBL only in the presence of GSH or S-methyl-GSH but not glucuronate. Uptake was osmotically sensitive and saturable for VBL (Km — 1.5 jM at 5 mM GSH) and GSH (half-maximum stimulation of ATP-dependent VBL uptake at —2 mM). Furthermore, the low inhibitory effect of VBL on Mrp2-mediated ATP- dependent [3H]LTC4 uptake was potentiated by GSH in a concentration-dependent fashion. Sf9-Mrp2 membrane vesicles also exhibited osmotic-dependent uptake of [3H]GSH. Uptake of [3H]GSH was independent of ATP and inhibited by LTC4, E217SG, and MK571, but only in the presence of ATP. Although GSH stimulated Mrp2-mediated [3H]VBL uptake, Mrp2-mediated [3H]GSH uptake was not affected by VBL.

In view of a putative function for Mrp2 in the kidney, we investigated whether the prototypic renal organic anion PAH is a substrate for Mrp2 (chapter 5).

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Membrane vesicles of Sf9-Mrp2 cells exhibited ATP-dependent uptake of [14C]PAH. Initial uptake rates in Sf9-Mrp2 membrane vesicles were —2-fold increased as compared to rates in membrane vesicles from mock-infected cells. Mrp2-mediated ATP-dependent [14C]PAH uptake was saturable (Km —2 mM) and susceptible to inhibition by LTC4 with a Ki value of —1 jM , which is in close proximity with its Km value. Mrp2-mediated ATP-dependent uptake of [14C]PAH was strongly inhibited by E217SG, whereas PAH inhibited ATP-dependent [3H]E217SG uptake only modestly. Fluorescein-methotrexate, lucifer yellow, FURA-AM, and probenecid also inhibited ATP-dependent uptake of [14C]PAH. These results indicate that Mrp2 mediates ATP-dependent PAH transport and might contribute to the renal excretion of PAH.

General conclusions & future perspectives

A prerequisite of this thesis was to overexpress rabbit Mrp2 in a cell system for functional studies. This appeared to be easier said than done, and took much longer than anticipated. Initial transfection experiments were performed with various cell lines, including MDCK cells. Throughout culturing, selected clones started to detach from the culture dish. W e hypothesized that overexpression of Mrp2 might result in a substantial loss of an essential compound from these cells. Looking back, GSH might have been this compound. Subsequently, we moved from constitutive expression to inducible expression using the tetracyclineregulatory expression system. Indeed, we obtained several MDCK clones with tetracyclin-inducable expression of mrp2 mRNA, but when clones were cultured for longer than 48 hours, mRNA dropped to undetectable levels (R.A.M.H. van Aubel, not shown). Furthermore, immunoblot analysis did not show any expression of Mrp2. We finally sought for an alternative approach and came across the baculovirus expression system, which turned out to be successful. This system is nowadays easy to handle and does no longer require extensive purification of

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the virus, since the viral genome with the cDNA of interest is isolated from a purified E. coli clone. In insect (Sf9) cells infected with a recombinant baculovirus, Mrp2 was expressed at high levels. The advantage of this system is the possibility of culturing large volumes of infected Sf9 cells necessary for isolation of membrane vesicles. A disadvantage, however, is that expression levels of Mrp2 may vary among different cultures, but this is not different from any other transient expression system. Nonetheless, a cell line stably transfected with an mrp2 cDNA is desirable.

What have we learned from the studies in this thesis? First of all, we obtained conclusive evidence in chapter 2 that our rabbit clone represents the true rabbit ortholog of rat Mrp2. Additional transport studies in membrane vesicles will answer the question whether the putative substrates fluorescein-methotrexate, lucifer yellow, and phenolphthalein glucuronide indeed are transported by Mrp2. Coinciding with the results in chapter 2, we show in chapter 3 that like rat Mrp2, rabbit Mrp2 is expressed at the apical membrane of hepatocytes and renal proximal tubular cells. The finding that Mrp2 is also present in the apical membrane of small intestinal villi not only suggests that Mrp2 can transport compounds into the intestinal lumen, but can also form a barrier for absorption of compounds. In chapter 4, we show that Mrp2 requires GSH for ATP-dependent transport of the cationic drug VBL. A proposed mechanism for GSH on Mrp2- mediated VBL transport is cotransport of the two compounds. However, such a mechanism is not supported by the finding that VBL did not affect GSH uptake into Sf9-Mrp2 membrane vesicles. On the other hand, it cannot be excluded that stimulation by VBL is undetectable above background of GSH uptake. At this stage, the role of GSH on drug transport by MRP2 (and MRP1) is far from being resolved. Perhaps, reconstitution of MRP2 into planar lipid bilayers might give more insight into this mechanism. Finally, in chapter 5, PAH was quite surprisingly identified as an Mrp2 substrate. The question remains whether Mrp2 plays an important role in the renal excretion of PAH. Therefore, comparative

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studies with wildtype and GY/TR- rats need to be further expanded and the involvement of other apical organic anion transporters should be examined.

An important part in the study of Mrp2 is its ATPase activity and the effect of anionic and cationic drugs. In a preliminary study, E217SG-induced ATPase activity was found in alkaline-stripped membranes of Sf9 cells expressing Mrp2 (R.A.M.H. van Aubel, not shown). Unfortunately, this result was difficult to reproduce for reasons yet unknown and therefore optimizing the purification of Mrp2 from crude Sf9 membranes will be necessary. In this thesis, we characterized the transport activities of wildtype Mrp2. In future, it will be interesting to investigate how truncation of the amino terminus, which is the site involved in transport of (at least) LTC4, affects Mrp2 ATPase activity. Furthermore, how does truncation of Mrp2 affects GSH transport and GSH-stimulated ATP- dependent vinblastine transport? Solving these questions will enhance our insight in the transport activities of Mrp2.

The specific polyclonal antibodies described in chapter 2 (k51mrp2) and 3 (GP8) enable future investigations on the effect of various pathophysiological conditions (ischaemia, drug-induced toxicity) on the expression level of Mrp2 in the kidney. In parallel, the response of the GY/TR- kidney to such conditions can be investigated. Despite the availability of the GY/TR- rat, it might be desirable to obtain mice homozygous for a disrupted mrp2 gene, since long-term breeding of the mutant rats might have resulted in acquired compensatory mechanisms. Second, such an mrp2 knock-out mouse could be cross-breeded with, for example, mdr1a (-/-) mdr1b (-/-) double-knockouts, which are homozygous for disrupted genes encoding Pgp drug-efflux pumps. Like Mrp2, Pgps are confined to the apical membrane of hepatocytes (Mdr1a/b), renal proximal tubules (Mdr1a/b), and small intestinal villi (Mdr1a). An mdr1a (-/-) mdr1b (-/-) mrp2 (-/-) triple knockout mouse therefore should contribute to the knowledge of these drug transporters in the renal and intestinal excretion of xenobiotics.

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Samenvatting en Conclusies(Summary and Conclusions in Dutch)

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Samenvatting en Conclusies

Samenvatting

Een belangrijke functie van de nier is het uitscheiden van lichaamseigen en lichaamsvreemde stoffen met de urine. De nier functioneert op de eerste plaats als een filter, maar beschikt daarnaast over zogenaamde transporters of transportsystemen, die in het algemeen specifiek zijn voor anionische (negatief- geladen) of kationische (positief-geladen) stoffen. In het eerste (proximale) deel van de niertubuli (nierbuisjes), waarvan er ongeveer één miljoen per menselijke nier aanwezig zijn, worden anionische en kationische stoffen uit het bloed in de tubuluscellen opgenomen en vervolgens uitgescheiden naar het lumen waarin zich de voorurine bevindt. Anionische stoffen worden via een zogenaamd actief transportsysteem vanuit het bloed, door de celmembraan, opgenomen in de proximale tubuluscellen. Daar de cel van binnen negatiever geladen is dan erbuiten, moeten anionische stoffen opgenomen worden tegen een electrochemische gradient. Aangezien een dergelijk transport energie vereist, wordt van een actief transportsysteem gesproken. In het onderzoek van de afgelopen 20 jaar heeft vooral dit opnamesysteem centraal gestaan, waarbij de anionische verbinding para-aminohippuraat (PAH) als modelstof is gebruikt. Veel minder onderzoek is verricht naar het transport vanuit de tubuluscel, via de luminale of apicale membraan, naar de voorurine. Daarentegen is er de laatste jaren wel veel bekend geworden over een organisch-aniontransportsysteem dat zich in de apicale (of canaliculaire) membraan van de levercel bevindt. Dit transportsysteem, canaliculaire multispecifieke organisch-aniontransporter (cMOAT) genaamd, bewerkstelligt het actieve (ATP-afhankelijke) transport van anionische stoffen van de levercel naar de gal. In de rat is cMOAT uitvoerig bestudeerd, mede door het bestaan van twee bijzondere rattenstammen met een defect in cMOAT. Deze ratten hopen in de lever het glucuronideconjugaat van de galkleurstof bilirubine op, een stof die in gezonde ratten goed getransporteerd wordt door cMOAT.

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Het erfelijke materiaal (gen) waarin de gegevens zijn opgeslagen voor het cMOAT- eiwit, is ontdekt naar aanleiding van resultaten uit kankeronderzoek. Een groot probleem bij de behandeling van tumoren met chemotherapie is het op den duur resistent worden van de tumor tegen de gebruikte cytostatica. Anders gezegd, de tumor heeft geen last meer van de aanwezigheid van deze celdodende stof en kan dus verder groeien en uitzaaien. Deze resistentie wordt ondermeer veroorzaakt doordat veel tumoren gedurende de behandeling een transportsysteem aanmaken dat de cytostatica de tumorcel uitpompt. Tot nu toe zijn twee van deze transportsystemen geïdentificeerd, P-glycoproteïne (Pgp) en multidrug-resistance- protein (MRP) genaamd. Opmerkelijk was dat MRP niet alleen cytostatica (voornamelijk kationische verbindingen) transporteert maar, vergelijkbaar met cMOAT, vooral ook anionische verbindingen. Met de kennis over het MRP-gen uit de mens, heeft men vervolgens het cmoat/cMOAT-gen geïsoleerd uit respectievelijk de rat en de mens. Aangezien cMOAT en MRP erg veel op elkaar lijken en aangezien er bovendien nieuwe MRP-genen (MRP3 t/m MRP6) zijn gevonden, worden MRP en cMOAT nu respectievelijk, MRP1 en MRP2 genoemd. Met de isolatie van het MRP2-gen kon worden aangetoond dat dit transportsysteem niet alleen aanwezig is in de apicale membraan van de levercel maar ook de proximale niertubuluscel.

In 1995 werd op de afdeling Celfysiologie in samenwerking met de afdeling Farmacologie een konijnengen gevonden dat grote overeenkomst vertoonde met het mrp2-gen uit de rat. In dit proefschrift hebben we allereerst een celsysteem ontwikkeld om de transportkarakteristieken van het konijnen-Mrp2 te onderzoeken om vervolgens met dit systeem een mogelijke rol te bepalen voor Mrp2 in de nier. Hieronder volgt een uiteenzetting van de inhoud van de afzonderlijke hoofdstukken. Na een algemene inleiding over transportsystemen in de nier voor anionische verbindingen in hoofdstuk 1, beschrijven we in hoofdstuk 2 de ontwikkeling van het celsysteem waarmee Mrp2 is bestudeerd. Om dit celsysteem te verkrijgen hebben we gekweekte cellen afkomstig van ovariumweefsel van de mot (Spodoptera frugiperda- ofwel Sf9-cellen), zodanig genetisch gemodificeerd

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dat ze konijnen-Mrp2 produceren. Daarvoor zijn deze cellen geïnfecteerd met een baculovirus waarin het mrp2-gen is aangebracht. Wanneer het virus de cel is binnengedrongen, wordt het ontmanteld en komt het mrp2-gen beschikbaar om in de cel te dienen als matrijs voor de synthese van het Mrp2-eiwit. In hoofdstuk 2 laten we zien dat het bereikte expressieniveau van Mrp2 in de baculovirus- geïnfecteerde Sf9-cellen ongeveer 4-maal hoger was dan het expressieniveau in levercellen en proximale niertubuli. Voor transportstudies hebben we gebruik gemaakt van een uit de geïnfecteerde cellen geïsoleerde (Mrp2-bevattende) membraanfractie, de zogenaamde membraanvesicles. Ter controle hebben we altijd membraanvesicles uit 'Mrp2-loze' Sf9-cellen gebruikt. Bovendien voegden we ATP aan de membraanvesicles toe, aangevuld met een zogenaamd ATP- regenerend systeem dat het door Mrp2-verbruikte ATP opnieuw synthetiseert. We hebben laten zien dat konijnen-Mrp2 de twee lichaamseigen anionische stoffen leukotrieen-C4 (LTC4) en estradiol-17-S-D-glucuronide (E2I 7SG) ATP-afhankelijk transporteert. Bij een ATP-concentratie van ongeveer 0.6 mM was het Mrp2- gemedieerde transport van E2I 7SG half-maximaal. Tenslotte hebben we laten zien dat het ATP-afhankelijke E2I 7SG- transport geremd kon worden door LTC4, MK571, cyclosporine A, methotrexaat (MTX), fluoresceïne-methotrexaat (FL-MTX) en het glucuronideconjugaat van fenolftaleïne. Daarentegen hadden fluoresceïne (FL) en het glucuronideconjugaat van naftol en nitrofenol geen effect. In hoofdstuk 3 laten we de lokalisatie zien van Mrp2 in de nier, lever en dunne darm van het konijn. Daartoe hebben we plakjes (coupes) van deze weefsels bekeken na behandeling met een antilichaam, specifiek gericht tegen Mrp2. We laten niet alleen de lokalisering van Mrp2 zien in de apicale membraan van levercellen en proximale tubuluscellen, maar ook in de top van de vlokken van de dunne darm. In hoofdstuk 4 hebben we onderzocht of konijnen-Mrp2, in analogie met MRP1, het cytostaticum vinblastine kan transporteren. Door gebruik te maken van de eerdergenoemde geïsoleerde membraanvesicles, werd duidelijk dat Mrp2 vinblastine alléén transporteert in aanwezigheid van gereduceerd glutathion (GSH), een belangrijk bestanddeel van elke cel. Andere stoffen, zoals S-methyl-

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GSH en glucuronaat, konden de rol van GSH niet overnemen. Half-maximaal Mrp2-gemedieerd transport van vinblastine werd bereikt bij een GSH-concentratie van ongeveer 2 mM. Bij een vinblastineconcentratie van 1.5 ¡j M (in aanwezigheid van 5 mM GSH) was het Mrp2-gemedieerde vinblastinetransport half-maximaal. Terwijl GSH kennelijk essentieel is voor Mrp2-gemedieerd vinblastinetransport, bleek vinblastine niet noodzakelijk te zijn voor Mrp2-gemedieerd GSH-transport. Bovendien werd GSH, in tegenstelling tot vinblastine, ATP-onafhankelijk getransporteerd door Mrp2. Het passieve Mrp2-gemedieerde GSH-transport kon echter alléén geremd worden door MK571 en de Mrp2-substraten, LTC4 en E217SG, in de aanwezigheid van ATP. In hoofdstuk 5 is tenslotte aangetoond dat Mrp2 de modelverbinding PAH op een ATP-afhankelijke wijze transporteert. Dit suggereert een rol voor Mrp2 in de renale uitscheiding van deze stof. Bij een concentratie van ongeveer 2 mM was het Mrp2-gemedieerde transport van PAH half-maximaal. Opvallend was dat de membraanvesicles zonder Mrp2 ook een behoorlijk ATP-afhankelijk PAH-transport vertonen. De aanwezigheid van Mrp2 zorgde uiteindelijk maar voor een verdubbeling van de initiële transportsnelheid. Terwijl E217SG een sterke remmer was van het Mrp2-gemedieerde PAH-transport, bleek PAH een zwakke remmer van Mrp2-gemedieerd E217£G-transport te zijn.

Conclusies

Wat zijn nu de belangrijkste conclusies die we uit dit proefschrift kunnen trekken? Op de eerste plaats is aangetoond dat het product van het geïsoleerde konijnengen inderdaad een transporteiwit is, identiek aan Mrp2 van de rat (Hoofdstukken 2 en 4). We hebben een celsysteem (Sf9) ontwikkeld dat op zichzelf niet nieuw is, maar wel door ons als eerste is gebruikt voor het karakteriseren van Mrp2. Het celsysteem, zoals beschreven in dit proefschrift, kan uiteindelijk dienen voor de screening van een hele reeks stoffen met als doel te bepalen of deze via Mrp2 getransporteerd worden. Met deze benadering hebben we laten zien dat de

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modelverbinding PAH een substraat is van Mrp2 (Hoofdstuk 5). Het is echter nog onduidelijk wat de bijdrage is van Mrp2 aan de renale uitscheiding van PAH. Studies met Mrp2-deficiënte en normale ratten kunnen daar meer informatie over verschaffen. Het beschreven celsysteem leent zich verder ook uitstekend om in de toekomst gemuteerde Mrp2-eiwitten te produceren en het effect te onderzoeken van aminozuurveranderingen op de transportfunctie.

In overeenstemming met de uitscheidingsfunctie van Mrp2 is aangetoond dat het eiwit zich in de apicale membraan van de levercel, de proximale niertubulus en de top van de vlokken van de dunne darm bevindt (Hoofdstuk 3). In alle drie de weefsels is dit dé lokatie voor een transporter die tot taak heeft potentieel giftige stoffen uit het lichaam te verwijderen. Bovendien kan Mrp2 in de dunne darm een rol spelen bij het vormen van een barrière, waardoor de biologische beschikbaarheid van bepaalde stoffen laag kan zijn. De in hoofdstuk 3 beschreven Mrp2-specifieke polyclonale antilichamen kunnen verder gebruikt worden om de invloed op het expressieniveau van Mrp2 te onderzoeken van verschillende pathofysiologische omstandigheden, zoals zuurstoftekort (ischemie) en toxische nierschade. Ondanks de beschikbaarheid van Mrp2-deficiënte ratten zou het wenselijk zijn om in de toekomst een Mrp2-deficiënte muizenstam te ontwikkelen. Een dergelijke Mrp2-knockout-muis zou gekruist kunnen worden met de reeds beschikbare P-glycoproteïne-deficiënte muis. P-glycoproteïne bevindt zich namelijk eveneens in de apicale membraan van onder meer lever-, darm- en niercellen. De uitschakeling van beide transporteiwitten zou meer informatie kunnen verschaffen over hun rol in de renale en intestinale uitscheiding van lichaamsvreemde stoffen.

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List of Publications

Deen PMT, Croes H, van Aubel RAMH, Ginsel LA, and CH van Os (1995) Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing. J. Clin. Invest. 95: 2291-2296.

Deen PMT, van Aubel RAMH, van Lieburg AF, and CH van Os (1996) Urinary content of

aquaporin 1 and 2 in nephrogenic diabetes insipidus. J. Am. Soc. Nephrol. 7: 836-841. van Kuijck MA, van Aubel RAMH, Busch AE, Lang F, Russel FGM, Bindels RJM, van Os CH, and

PMT Deen (1996) Molecular cloning and expression of a cyclic AMP-activated chloride conductance regulator: A novel ATP-binding cassette transporter. Proc. Natl. Acad. Sci. USA 93: 5401-5406.

van Aubel RAMH, van Kuijck MA, Koenderink JB, Deen PMT, van Os CH, and FGM Russel (1998)

Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance-associated protein Mrp2 expressed in insect cells. Mol. Pharmacol. 53: 1062-1067.

van Aubel RAMH, Koenderink JB, Peters JGP, Deen PMT, van Os CH, and FGM Russel (1998) Involvement of the multidrug resistance-associated protein 2 (Mrp2) in the renal excretion of drug glucuronide conjugates. J. Am. Soc. Nephrol. 9: A305 (Annual Meeting of the American Society of Nephrology, October 1998, Philadelphia, USA).

van Aubel RAMH, Koenderink JB, Peters JGP, van Os CH, and FGM Russel (1999) Mechanism and interaction of vinblastine and reduced glutathione transport in membrane vesicles by the rabbit multidrug resistance protein Mrp2 expressed in insect cells. Mol. Pharmacol. 56: 714-719.

Masereeuw R, Terlouw SA, van Aubel RAMH, Russel FGM, and DS Miller (2000). Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule. Mol. Pharmacol. 57: 59-67.

van Aubel RAMH, Masereeuw R, and FGM Russel (2000). Molecular pharmacology of renal organic anion transporters. Am. J. Physiol. (Renal Physiol.), in press.

van Aubel RAMH, Peters JGP, Masereeuw R, van Os CH, and FGM Russel (2000) The multidrug resistance protein Mrp2 mediates ATP-dependent transport of the classical organic anion p-aminohippurate. Submitted.

van Aubel RAMH, Hartog A, Bindels RJM, van Os CH, and FGM Russel (2000) Expression and

immunolocalization of multidrug resistance protein 2 in rabbit small intestine. Submitted.

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Dankwoord

Zonder de bijzondere bijdrage van een aantal mensen, zou ik dit proefschrift nooit hebben kunnen schrijven. Vanwege de 'moleculaire' aard van mijn werkzaamheden, zou de start van mijn AIO-tijd plaatsvinden bij de afdeling Celfysiologie in het Trigon-gebouw. Uitgezonderd enkele verhuizingen naar een andere werkplek, heb ik de labs van deze groep uiteindelijk nooit verlaten. Aangezien de groep in de loop van mijn promotie nogal van samenstelling is veranderd, is het een onmogelijke taak om iedereen individueel de revue te laten passeren. Hoe dan ook, ik dank iedereen die bij Celfysiologie heeft gewerkt of nog werkt voor een goede samenwerking en prettige werksfeer. In het bijzonder dank ik de 'vaste staf' Prof. dr. Carel van Os, Dr. René Bindels en Dr. Peter Deen voor de steun, een kritische blik en bovendien nuttige tips bij de werkbesprekingen. Ik dank ook alle medewerkers van de afdeling Farmacologie & Toxicologie, in het bijzonder, Prof. dr. Paul Smits, Dr. Roos Masereeuw, Janny Peters en Pascal Smeets. Janny, je bent een enorme hulp geweest bij de transportstudies en vesicle isolaties. De 'credits' voor hoofdstuk 5 zijn wat dat betreft eigenlijk voor jou bestemd. Ik heb bovendien ontzag voor je -in mijn ogen- ingewikkelde Excel- schema's die ik wel nooit zal begrijpen. Pascal wens ik veel succes met zijn promotie-onderzoek. Last but not least, gaat mijn bewondering uit naar mijn eerste promotor Prof. dr. Frans Russel. Ik heb in die vier jaar erg veel opgestoken van jouw kennis over transportstudies en transportprocessen. Ik heb bovendien erg veel plezier beleeft aan onze tripjes naar Ascona (daar kan het trouwens erg hard regenen), Halle, Philadelphia en Gosau. Volgens mij ben ik je eerste promovendus geweest, waarvoor je een cursus moest doorlopen voordat je kon plaatsen waar ik het over had, wanneer ik je kamer kwam binnenstormen en klaagde over problemen met kloneringen en transfecties. Ten slotte kan ik ook niet om de vakgroep Biochemie (FMW) heen zonder een woord van dank. Zonder de bijzondere bijdrage van de 'ATPase' tak van deze groep zouden de resultaten misschien nog lang zijn uitgebleven. Prof. dr. Jan-Joep de Pont, Drs. Harm

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Hermsen, Herman Swarts en, in het bijzonder, Drs. Jan 'baculo' Koenderink dank ik voor hun bijdrage aan dit proefschrift. Jan, ik heb met veel plezier met je samengewerkt en hoop dat in de toekomst nog vaker te kunnen doen. Jouw co- auteurschap op twee van mijn artikelen onderstreept jouw bijdrage des te meer.

Saskia, mijn vrouw en maatje. Sas, in moeilijke tijden wanneer mijn motivatie wegzakte, nam je mijn aandacht weg van het werk, waardoor ik er weer sterker uitkwam. Als jij me bij aanvang van dit project had verteld hoe mijn leven aan het einde ervan zou hebben uitgezien, dan zou ik je voor gek hebben verklaard. We zijn eerst getrouwd en hebben daarna het mooiste mannetje van de wereld gekregen. Ik geniet er iedere dag van. Dankjewel.

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Curriculum Vitae

Rémon van Aubel werd geboren op 23 juli 1970 te Schaesberg. In juni 1987 en juni 1989 behaalde hij het achtereenvolgens het HAVO- en het VWO-diploma aan het Eijckhagencollege te Landgraaf. In september 1989 werd begonnen met de studie biologie aan de toenmalige Faculteit Wiskunde en Natuurwetenschappen van de Katholieke Universiteit Nijmegen. In het kader van deze studie werd een hoofdvakstage gelopen bij de afdeling Medische Microbiologie/Virologie (Dr. W. Melchers) en een bijvakstage bij de afdelingen Celfysiologie (Prof. dr. C.H. van Os) en Dierfysiologie (Prof. dr. S.E. Wendelaar Bonga). In januari 1995 behaalde hij het doctoraaldiploma. Aansluitend startte hij als Assistent-In-Opleiding (AIO), in dienst van de Faculteit der Medische Wetenschappen, bij de afdeling Farmacologie & Toxicologie. Hier voerde hij onder leiding van Prof. dr. F.G.M. Russel en in nauwe samenwerking met Prof. dr. C.H. van Os, Dr. P.M.T. Deen en Dr. R.J.M. Bindels van de afdeling Celfysiologie, het in dit proefschrift beschreven onderzoek uit. Tijdens dit promotieonderzoek bezocht hij het European Symposium on Renal and Hepatic Transport (Halle, Duitsland, oktober 1997), de 31th annual meeting of the American Society of Nephrology (Philadelphia, USA, oktober 1998), de 2nd FEBS Advanced Lecture Course on ATP-binding cassette proteins: from multidrug resistance to genetic disease (Gosau, Oostenrijk, februari 1999) en de International PharmaConference on Membrane Transporters (Ascona, Zwitserland, augustus 1999).

Sinds 1 februari 1999 is hij werkzaam als post-doc-onderzoeker, opnieuw bij de afdeling Farmacologie & Toxicologie. Hij is getrouwd met Saskia Nies en vader van Hidde van Aubel.

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