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Nijmegen
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Multidrug Resistance Protein 2
Transport Properties o f a Drug Efflux Pump
R.A.M.H. van Aubel
Transport Properties of a Drug Efflux Pump
R.A.M.H. van Aubel. Thesis University of Nijmegen
-With Ref.- W ith 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
Transport Properties of a Drug Efflux Pump
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)
Transport Properties o f a Drug Efflux Pump
Contents
Abbreviations
9
Chapter 1
Introduction: molecular pharmacology of renal organic anion transporters
11
Chapter 2
Adenosine triphosphate-dependent transport of anionic conjugates by the
rabbit multidrug resistance protein Mrp2 expressed in insect cells
49
Chapter 3
Immunolocalization of the multidrug resistance protein Mrp2 in rabbit small
intestine, liver, and kidney
69
Chapter 4
Mechanisms and interaction of vinblastine and reduced glutathione transport
in membrane vesicles by the rabbit multidrug resistance protein Mrp2
expressed in insect cells
87
Chapter 5
The multidrug resistance protein Mrp2 mediates ATP-dependent transport of
the classical organic anion p-aminohippurate
105
Chapter 6
Summary, general conclusions and future perspectives
119
Samenvatting en conclusies
129
List of publications
137
Dankwoord
139
Curriculum Vitae
141
Abbreviations
ABC
ATP-binding cassette
ANOVA
analysis of variance
5'-AMP
adenosine 5'-monophosphate
ATP
adenosine triphosphate
BBM
brush-border membrane
BLM
basolateral membrane
BSA
bovine serum albumin
CDDP
c/s-diamminedichloroplatin
cD N A
complementary D N A
CFTR
cystic fibrosis transmembrane conductance regulator
cM O AT
canalicular multispecific organic anion transporter
CsA
cyclosporin A
DM SO
dimethylsulphoxide
DNP-SG
5-(dinitrophenyl)-glucuronide
DTT
dithiothreitol
E2175G
17fi-estradiol-17-5-D-glucuronide
EDTA
ethylenediamine tetraacetate
EHBR
Eisai hyperbilirubinemic rat (Mrp2-deficient Sprague Dawley rat)
FL
fluorescein
FL-MTX
fluorescein-methotrexate
FURA-AM
fura-2 acetoxy methyl ester
GSH
reduced glutathione
GSSG
oxidized glutathione
GY/TR-
Groningen yellow/ Transport deficient rat (Mrp2-deficient Wistar rat)
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid
IgG
immunoglobulin G
kb
kilobases
kDa
kilodaltons
Ki
inhibition constant
Km
Michaelis-Menten constant
LTC4
leukotriene C4
LY
lucifer yellow
mAb
monoclonal antibody
MDCKII
type II (proximal tubule derived) kidney cell line of Madin-Darby canine
MDR
multidrug resistance
MK571
3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethyl-amino-3oxopropyl)-thio}-methyl]thio)propanoic acid
MOI
multiplicity of infection
MRP
multidrug resistance protein
MTX
methotrexate
OA
organic anion
PAGE
polyacrylamide gel electrophoresis
PAH
para-aminohippurate
PBS
phosphate-buffered saline
Pgp
P-glycoprotein
PLP
periodate lysine paraformaldehyde
PMSF
phenyl methyl sulphonyl fluoride
PNGase F
N-glycosidase F
PT
Proximal tubule
SD
standard deviation
SDS
sodium dodecyl sulphate
SE
standard error of the mean
Sf9
Spodoptera frugiperda cells
TBS
tris-buffered saline
TS
tris sucrose
Tris
Tris(hydroxymethyl)aminomethane
Tween 20
polyoxyethylene sorbitan monolaurate
VBL
vinblastine
VCR
vincristine
Vmax
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. Russel
Depts. 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......................................................................................... 21
Organic anion transporter Oat1 (PAH/dicarboxylate exchanger).......................... 22
Oat2 and Oat3/OAT3......................................................................................... 23
Sulfate/anion exchanger Sat1 ...............................................................................24
Multidrug resistance protein M RP1......................................................................24
MRP3, MRP4, MRP5 and MRP6...........................................................................25
Intracellular disposition of anionic drugs........................................................................26
Apical transport systems................................................................................................ 28
MRP2 ................................................................................................................ 29
Organic anion transport polypeptides Oatp1 and Oatp3...................................... 31
Organic anion transporters Oat-k1 and Oat-k2..................................................... 31
Na+/nucleoside cotransporters CNT1 and CNT2.................................................. 32
H+/peptide cotransporters PEPT1 and PEPT2....................................................... 33
Conclusive remarks....................................................................................................... 33
Aim 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
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
anti
inflammatory 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. W hile 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 1
Molecular characteristics of organic anion transporters in the kidneya
Name
Alternative
name
Species
Acc. No.
Chromosome
localization
OAT1
human
60
2.4
rat
flounder
mouse
AF097490/AB009697/
AF057039/AF104038
AB004559/AF008221
Z97028
U52842
11q11.7
Oat1
Oat1
Oat1
hPAHT/
hROATI
ROAT1
fRO A T
NKT/m OAT
19
60
60
60
Oat2
NLT
rat
L27651
OAT3
Oat3
Oat3
Roct
human
mouse
rat
AF097491
AF078869
AB017446
rat
L23413
Sat1
11q11.7
19
Mrb
(kDa)
mRNA
(kb)
TM
Tissue
Distribution
Nephron
Distribution
Membrane
Localization
References
12
k
PT
B LM
[59,105,150,152]
2.4
nd
2.5
12
12
11
k
nd
k
PT (S2)
nd
PT
B LM
nd
nd
[166,176,186,191]
[205]
[89,103]
60
2.1
12
k, li
nd
nd
[165,172]
62
62
3.0
2.1
12
12
k
k
k, li, b, e
nd
PT
nd
nd
nd
nd
[150]
75
3.7
12
k, li, b, m
PT (S1-S3)
B LM
[9,73]
[13]
[88]
MRP1
Mrp1
M RP
human
mouse
L05628
AF022908
16p13.12-13
171
171
6.5
6.5
18
18
ubiquitous
ubiquitous
nd
LH, C C D
nd
B LM
[20,39,213]
[174,201,203]
M RP2
cM O AT/
cM RP
human
NM_000392/
U63970/ U49248/
X96395
L49379/ D86086/
X96393
Z49144
10q24
175
6.5
18
k, li, si, n
nd
nd
[82,181]
175
6.5
18
k, li, si
PT (S1-S3)
BBM
[75]
175
6.5
18
k, li, si
PT (S1-S3)
BBM
[194], unpublished
17q21.3
170
6.5
18
k(?), li, si, c, lu,
nd
B LM c
[42,78,82,84,188]
Mrp2
rat
Mrp2
rabbit
M RP3
MOAT-D/
c M O A T2
human
Mrp3
M LP2
rat
Y17151/ AF104943
NM_003786/
AF085690/ AF009670
AB010887/ AF072816
M RP4
M OAT-B
human
U83660/ AF071202
M RP5
MOAT-C/
SMRP/
pABC11
human
Mrp5
M RP6
Mrp6
Mrp6
MOAT-E
MLP1
p
170
6.5
18
k, si, c, lu
nd
nd
[57]
13q31-32
150
6.0
12
(k, lu, bl, gb)d
ubiquitous6
nd
nd
[82]
[90]
3q27
160
nd
12
ubiquitous
nd
B LM
[5,82,118,202]
mouse
U83661/ AF104942/
AB005659/AB019002
/AF146074
AB019003
160
nd
12
nd
nd
nd
unpublished
human
rat
mouse
AF076622
AB010466/ U73038
AB028737
16p13
165
165
165
6.0
6.0
nd
18
18
18
k, li
k, li, du
nd
nd
nd
nd
nd
B LM c
nd
[6,83]
[57,107]
unpublished
Name
Alternative
name
Species
Acc. No.
Chromosome
localization
Tissue
Distribution
Nephron
Distribution
Membrane
Localization
References
O atp l
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)/ C C D
BBM
[114]
human
rat
15q25-26
p ig
U62968
U10279
AF009673
71
71
71
3.4
3.0
nd
14
14
14
k, si, li
k, si, li
nd
nd
nd
nd
nd
nd
nd
[153]
[38,60]
[137]
human
rat
mouse
U84392
U25055
AF079853
15q15
71
71
71
4.5
3.5
nd
14
14
14
ubiquitous
k, si, li
nd
nd
nd
nd
nd
nd
nd
[154,198]
[18,38]
unpublished
PEPT1
human
13q33-34
78
3.3
12
k, si, li, pl
nd
nd
[95]
PepT1
PepT1
PepT1
rat
mouse
rabbit
AF043233/ U13173/
U21936
D50306/ D50664
Y07561
U06467/ U13707
78
78
78
3.0
nd
2.9
12
12
12
k, si
nd
k, si, li, b
PT (S1-S2)
nd
cortex
BBM
nd
nd
[126,159,169]
unpublished
[11,37]
PEPT2
PepT2
PepT2
human
rat
rabbit
S78203
D63149
U32507
3q13.3-q21
78
78
78
4.2
4.0
4.8
12
12
12
k, b
k, b, lu
k, b, lu, li
nd
PT (S3)
nd
nd
BBM
nd
[151]
[160,169,197]
CNT1
Cnt1
Cnt1
CNT2
Cnt2
Cnt2
cNT1
hSPNT1
SPN T
Mrb
(kDa)
mRNA
(kb)
TM
[10]
“Abbreviations: (R)O AT: (renal) organic anion transporter; hPAH T: human PAH transporter; NKT: novel kidney transporter; NLT: novel liver transporter; Sat1 : sulfate anion
transporter; M RP: multidrug resistance protein; c M O A T : canalicular multispecific organic anion transporter; cM R P: canalicular M R P ; M LP: 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; B B M : 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; C C D : 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 2
Functional characteristics of organic anion transporters in the kidneya b
Name
transport
mechanism
Substrates (Km)
Inhibitors (Ki)
References
unconjugated_______________ conjugated
OAT 1
human
OA/
dicarboxylate
antiport
PAH (5 uM)
furosemide; indomethacin;
probenecid; urate; a-KG;
glutarate
[59,105,150]
Oat1
rat
OA/
dicarboxylate
antiport
PAH (14-70 uM);
salicylate; MTX; cAMP;
acetylsalicylate;
indomethacin; folate;
cG M P; PGE 2 ; urate; aKG; ochratoxin A
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; PGE 2 ; cAMP;
cG M P
[3,166,176,
187,191]
Oat2
rat
nd
(18 uM ); salicylate (90
uM ); acetylsalicylate
bumetanide; enalapril;
cefoperazone; cholate
[165]
Oat3
rat
nd
PAH (65 uM);
ochratoxin A (0.74 uM);
estrone-sulfate (2.3 uM);
cimetidine
BSP; probenecid; indocyanine
green; bumetanide; piroxicam;
furosemide; AZT; DlDS
[88]
Sat1
rat
OA/ sulfate
antiport
sulfate (320 uM);
oxalate
DIDS; oxalate; sulfate
[9,73]
MRP1
human
primary active
GSH; PAH
LTC4 (0.1 uM); E2 1 7 KG (1.5
uM); DNP-SG (3.6 uM);
bilirubin-glucuronide; GSSG
(93 uM); A FB 1-SG (0.2 uM);
PG A 1-SG; PG A 2-SG (1 uM);
etoposide-glucuronide; S(ethacryn ic acid)-glutathione
MK571 (0.6 uM); CsA (5 uM);
PSC833 (27 uM ); S-(decyl)glutathione (0.7uM); GS-DOX
(60/120 nM); GS-DAU
(70/200 nM); probenecid
[33,35,69,70,
76,92, 98,99,
101,145,149,
210]
MRP2
human
primary active
GSH; MTX
LTC4(1 uM ); E217KG (7 uM);
DNP-SG; PG A 1-SG;
bilirubin-glucuronide; S(ethacryn ic acid)-glutathione
[24,34]
Mrp2
rat
GSH; MTX (100 uM);
folate; pravastatin (170
uM ); BSP (31 uM ); Fluo3 (4 uM ); BQ123 (100
uM ); BQ485 (7 uM);
BQ518 (15 uM);
temocaprilat (100 uM);
cefpiramide; ceftriaxone
LTC4(1 uM ); E217SG (7 uM); MK571; CsA; GSH (20 mM)
LTD4(1.5 uM); N-acetyl- LTE4
(5.2 uM ); DNP-SG (0.1 8 uM);
bilirubin-glucuronide; GSSG
(111 uM ); p-nitrophenylglucuronide (20 uM); anaphthyl-^-D-glucuronide;
E3040-glucuronide
[2,24,63,65,
67,70, 79, 87,
106,113,130,
134-136,142,
194, 206]
Mrp2
rabbit
GSH
LTC4(1 uM ); E217SG
[192-194]
MRP3
Mrp3
human
rat
primary active
MTX
MTX
DNP-SG
E217KG (70 uM); E3040glucuronide
M RP4
human
primary active
PMEA; AZTM P
dipyridamole
[164]
MRP5
human
primary active
CM FDA; BCECF; FDA
sulfinpyrazone
[118]
Mrp6
rat
primary active
BQ123
18
PAH; lucifer yellow;
fluorescein-methotrexate;
phenolphthalein-glucuronide;
CsA, probenecid
E3040-glucuronide;
a-naphthyl-j3-D-glucuronide;
MTX
[84]
[58]
[1 0 7 ]
Name
transport
mechanism
Substrates (Km)
unconjugated
TC (27 uM ); BSP (1.5 uM);
aldosterone (15 nM);
cortisol (13 nM); ouabain
(17 mM); ochratoxin A
(17 uM ); thyroxine;
triiodo-L-thyronine;
enalapril (214 uM);
temocrapilat (47 uM)
Inhibitors (Ki)
conjugated
LTC4 (0.27 j j M ); DNP-SG
(408 ^M); E217ßG (3 ^M);
estrone-sulfate (4.5 >wM)
References
Oatpl
rat
OA/GSH
antiport
Oatp3
rat
nd
TC (18 uM ); thyroxine (5
uM ); triiodo-L-thyronine (7
uM )
Oat-k1
rat
nd
MTX (1 uM ); folate
MTX; BSP; probenecid; PAH;
furosemide; valproate; DIDS;
TCA; ibuprofen; flufenamate;
phenylbutzone; indomethacin
(1 mM); ketoprofen (2 mM)
[116,158]
Oat-k2
rat
nd
TC (10 ywM); MTX; PGE 2 ;
folate
MTX; BSP; probenecid; PAH;
furosemide; valproate; DIDS;
TCA; levofloxacin;
indomethacin; testosteron;
dexamethasone; 17^-estradiol;
ouabain
[114]
CNT1
Cnt1
human
rat
nucleoside/
Na+ symport
adenosine (15/26 uM);
thymidine (13 uM);
uridine (26/45); AZT (0.5
mM); dideoxycytidine (0.5
mM)
AZT; cytarabine;
dideoxycytidine; floxidine;
idoxuridine; thymidine;
cytidine; uridine
[36,60,153,
207,208]
CNT2
human
adenosine (8 uM); uridine
(40/80 uM ); inosine (5
uM )
formycin B, guanosine,
adenosine, uridine, inosine
[154,198]
adenosine (6 uM); inosine
(20 uM ); uridine (20 uM);
cladribine, thymidine (13
uM )
formycin B, guanosine,
uridine, inosine, thymidine
ampicillin (50 mM);
amoxicillin (13 mM);
cyclacillin (0.17 mM);
cephalexin (5 mM); cefadroxil
(2 mM); cephradine (9 mM);
cefdinir (12 mM); ceftibuten
(0.6 mM); cefixime (7 mM);
bestatin (0.5 mM); enalapril
(4.3 mM); captopril (9 mM); LVal-ACV (0.74 mM)
nucleoside/
Na+ symport
Cnt2
[32,41,64,71,
72,94,138]
[1]
rat
[18,162]
PEPT1
PepTI
PepTI
human
rat
rabbit
peptide/ H +
symport
glycylsarcosine (1.1 mM);
ALA (0.28 mM);
ceftibuten (0.3 mM);
cyclacillin (0.5 mM);
cephalexin; cefixime;
cefadroxil; L-Val-ACV (5
mM); L-Val-AZT; L-dopa-LPhe; /brmy/-Met-Leu-Phe
PEPT2
PepT2
PepT2
human
rat
rabbit
peptide/ H +
symport
glycylsarcosine (0.11
mM); ALA (0.23 mM);
cephalexin; bestatin; LVal-ACV
[4,31,43-45,
55,120,180,
182,183,200]
ampicillin (0.67 mM);
[31,43-45,183]
amoxicillin (0.18 mM);
cyclacillin (27 >wM); cephalexin
(50 juM); cefadroxil (3 juM);
cephradine (47 uM ); cefdinir
(20 mM); ceftibuten (1 mM);
cefixime (12 mM); bestatin (20
uM); L-Val-ACV (0.39 mM)
^Abbreviations: PAH: p-amminohippurate; OA: organic anion; a-KG: a-ketoglutarate; MTX: methotrexate; PGE 2, prostaglandin E2 ; BSP:
bromosulfophthalein; DIDS: 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid; LTC4 : leukotriene C 4 ; LTD 4 : leukotriene D 4 ; CsA: cyclosporin A;
E217£G: estradiol-17£-D-glucuronide; DNP-SG: dinitrophenyl-glutathione; GSSG, oxidized glutathione; GSH: reduced glutathione; A FB 1-SG, 5(aflatoxin B 1)-glutathione; E3040-glucuronide: 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole glucuronide; PMEA: 9-(2phosphonylmethoxyethyl)adenine; AZTMP: azidothymidine monophosphate; CM FDA: 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; PG A 2-SG: 5-(prostaglandin A 2)-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-ProThg-Leu).
bSubstrates mentioned for some transporters are indicative but not complete.
19
Chapter One
interstitium
lumen
Na+/K+-ATPase
K+
Z
^
i n
OAT1 a-KG2
OA-n
r
,
SO 4
OA'
OATP1
G SH
z n r
OAT-K1/K2
1/K2
z
SAT1
OA-
*• OA-
n
MRP2
OA-
CNT1/2
r z i
oa-— o
OA-?
oo
a Na+
-
SDCT2
Na+
dicarboxylates
N
Na+
nucleosides
PEPT1/2
HCO3
"
o
■4-----
—
H+
H e H t id 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 H C O 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 13acetate (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
22
putative
basolateral
Na+/dicarboxylate
exchanger
SDCT2,
as well
as
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/OAT3
Functional 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 Sat1
Expression 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 Sat1mediated 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 MRP1
The 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 C 4, 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 MRP6
Searching 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
overexpressing
human
MRPs
MRP3
have been
show
reported recently.
increased
efflux
of
MDCKII
cells
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 Mrp 6 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,
concentrations
3-5
PAH
times
the
and fluorescein
medium
accumulate
concentration
intracellularly at
[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 Stransferase, 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
mechanisms takes place
oxidation through cytochrome
predominantly
in kidney
P450-dependent
proximal
tubules
[102].
Glucuronide conjugates are formed through the enzymatic activity of UDPglucuronosyltransferase, 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 yglutamyltranspeptidase 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].
biotransformation
The
different
reactions
enzymes
involved
(y-glutamyltranspeptidase and
in
GSH-derived
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].
MRP2
The 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 M RP2 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 ATPbinding 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
methotrexate
and
leukotriene C4-sensitive efflux of fluorescein-
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
cefpiramide
[130],
a-naphtyl-^-D-glucuronide
the
quinolone
[28],
HSR-903
[131],
E3040-glucuronide
and
[179],
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 B 1 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 GSHstimulated 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 Oatp3
The organic anion-transporting polypeptide 1 (Oatp1) is a Na+- and ATPindependent 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 H C O 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-k2
Oat-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 M DCK 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 CNT2
The 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 brushborder 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 PEPT2
Peptide 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 highaffinity 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
[10,127,128,151,169,171,177,185].
in
This
brush-border
indicates
membrane
that
PEPT2
vesicles
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.
W e 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
1. Abe, T., M. Kakyo, H. Sakagami, T. Tokui, T. Nishio, M. Tanemoto, H. Nomura, S. C. Hebert, S. Matsuno, H.
Kondo, and H. Yawo. (1998). Molecular characterization and tissue distribution of a new organic anion
transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2. J. Biol.
Chem. 273: 22395-22401.
2. Akhteruzzaman, S., Y. Kato, A. Hisaka, and Y. Sugiyama. (1999). Primary active transport of peptidic endothelin
antagonists by rat hepatic canalicular membrane. J.Pharmacol.Exp.Ther. 575-581.
3. Apiwattanakul, N., T. Sekine, A. Chairoungdua, Y. Kanai, N. Nakajima, S. Sophasan, and H. Endou. (1999).
Transport properties of nonsteroidal anti-inflammatory drugs by organic anion transporter 1 expressed in Xenopus
laevis oocytes. Mol. Pharmacol. 55: 847-854.
4. Balimane, P. V., I. Tamai, A. Guo, T. Nakanishi, H. Kitada, F. H. Leibach, A. Tsuji, and P. J. Sinko. (1998). Direct
evidence for peptide transporter (PepT1)-mediated uptake of a nonpeptide prodrug, valacyclovir. Biochem.
Biophys. Res. Commun. 250: 246-251.
5. Belinsky, M. G., L. J. Bain, B. B. Balsara, J. R. Testa, and G. D. Kruh. (1998). Characterization of MOAT-C and
MOAT-D, new members of the MRP/cMOAT subfamily of transporter proteins. J. Natl. Cancer Inst. 90: 1735
1741.
6.
Belinsky, M. G. and G. D. Kruh. (1999). MOAT-E (ARA) is a full-length MRP/cMOAT subfamily transporter
expressed in kidney and liver. Br. J. Cancer 80: 1342-1349.
7. Bennett, W . M. (1996). Insights into chronic cyclosporine nephrotoxicity. Int. J. Clin. Pharmacol. Ther. 34: 515
519.
8.
Bergwerk, A. J., X. Shi, A. C. Ford, N. Kanai, E. Jacquemin, R. D. Burk, S. Bai, P. M. Novikoff, B. Stieger, P. J.
Meier, V. L. Schuster, and A. W . Wolkoff. (1996). Immunologic distribution of an organic anion transport protein
in rat liver and kidney. Am. J. Physiol. 271: G231-G238.
9. Bissig, M., B. Hagenbuch, B. Stieger, T. Koller, and P. Meier. (1994). Functional Expression Cloning of the
Canalicular Sulfate Transport System of Rat Hepatocytes. J. Biol. Chem. 3017-3021.
35
Chapter One
10. Boll, M., M. Herget, M. Wagener, W . M. Weber, D. Markovich, J. Biber, W . Clauss, H. Murer, and H. Daniel.
(1996). Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled
peptide transporter. Proc. Natl. Acad. Sci. USA 93: 284-289.
11. Boll, M., D. Markovich, W . M. Weber, H. Korte, H. Daniel, and H. Murer. (1994). Expression cloning of a
cDNA from rabbit small intestine related to proton-coupled transport of peptides, beta-lactam antibiotics and
ACE-inhibitors. Pflugers Arch. 429: 146-149.
12. Boyde, A., G. Capasso, and R. J. Unwin. (1998). Conventional and confocal epi-reflection and fluorescence
microscopy of the rat kidney in vivo. Exp. Nephrol. 6: 398-408.
13. Brady, K. P., H. Dushkin, D. Fornzler, T. Koike, F. Magner, H. Her, S. Gullans, G. V. Segre, R. M. Green, and D.
R. Beier. (1999). A novel putative transporter maps to the osteosclerosis (oc) mutation and is not expressed in the
oc mutant mouse. Genomics 56: 254-261.
14. Brown, D. Epithelial cell polarity: molecular mechanisms. In: Molecular Nephrology, kidney function in health
and disease, edited by D. Sclöndorff and J. V. Bonventre. New York: Marcel Dekker, Inc. (1995). p. 21-32.
15. Büchler, M., J. König, M. Brom, J. Kartenbeck, H. Spring, T. Horie, and D. Keppler. (1996). cDN A 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.
16. Campbell, J. A., N. M. Bass, and R. E. Kirsch. (1980). Immunohistological localization of ligandin in human
tissues. Cancer 45: 503-510.
17. Chatsudthipong, V. and W . H. Dantzler. (1992). PAH/alpha-KG countertransport stimulates PAH uptake and net
secretion in isolated rabbit renal tubules. Am. J. Physiol. 263: F384-91.
18. Che, M., D. F. Ortiz, and I. M. Arias. (1995). Primary structure and functional expression of a cDN A encoding
the bile canalicular, purine-specific Na( + )-nucleoside cotransporter. J. Biol. Chem. 270: 13596-13599.
19. Chen, X., H. Tsukaguchi, X.-Z. Chen, U. V. Berger, and M. A. Hediger. (1999). Molecular and functional analysis
of SDCT2, a novel rat sodium-dependent dicarboxylate transporter. J. Clin. Invest. 103: 1159-1168.
20. Cole, S. P. C., G. Bhardwaj, J. H. Gerlach, J. E. Mackie, C. E. Grant, K. C. Almquist, A. J. Stewart, E. U. Kurz, A.
M. Duncan, and R. G. Deeley. (1992). Overexpression of a transporter gene in a multidrug-resistant human lung
cancer cell line. Science 258: 1650-1654.
21. Cole, S. P. C. and R. G. Deeley. (1998). Multidrug resistance mediated by the ATP-binding cassette transport
protein MRP. Bioessays 20: 931-940.
22. Cole, S. P. C., K. E. Sparks, K. Fraser, D. W . Loe, C. E. Grant, G. M. Wilson, and R. G. Deeley. (1994).
Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells. Cancer Res. 54:
5902-5910.
23. Commandeur, J. N., G. J. Stijntjes, and N. P. Vermeulen. (1995). Enzymes and transport systems involved in the
formation and disposition of glutathione S-conjugates. Role in bioactivation and detoxication mechanisms of
xenobiotics. Pharmacol. Rev. 47: 271-330.
24. Cui, Y., J. König, U. Buchholz, H. Spring, I. Leier, and D. Keppler. (1999). Drug resistance and ATP-dependent
conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in
human and canine cells. Mol. Pharmacol. 55: 929-937.
25. Dahlmann, A., W . H. Dantzler, S. Silbernagl, and M. Gekle. (1998). Detailed mapping of ochratoxin A
reabsorption along the rat nephron in vivo: the nephrotoxin can be reabsorbed in all nephron segments by
different mechanisms. J. Pharmacol. Exp. Ther. 286: 157-162.
26. Daley Yates, P. T. and D. C. McBrien. (1984). Cisplatin metabolites in plasma, a study of their pharmacokinetics
and importance in the nephrotoxic and antitumour activity of cisplatin. Biochem.Pharmacol. 33: 3063-3070.
27. Daniel, H. and M. Herget. (1997). Cellular and molecular mechanisms of renal peptide transport. Am. J. Physiol.
273: F1-8.
28. de Vries, M. H., F. A. Redegeld, A. S. Koster, J. Noordhoek, J. G. de Haan, R. P. J. Oude Elferink, and P. L. M.
Jansen. (1989). Hepatic, intestinal and renal transport of 1-naphthol-beta-D-glucuronide in mutant rats with
hereditary-conjugated hyperbilirubinemia. Naunyn. Schmiedebergs. Arch. Pharmacol. 340: 588-592.
29. Dekant, W . and S. Vamvakas. (1996). Biotransformation and membrane transport in nephrotoxicity. Crit. Rev.
Toxicol. 26: 309-334.
36
30. Delacruz, L. and P. H. Bach. (1990). The role of ochratoxin A metabolism and biochemistry in animal and
human nephrotoxicity. J. Biopharm. Sci. 1: 277-304.
31. Doring, F., J. Walter, J. W ill, M. Focking, M. Boll, S. Amasheh, W . Clauss, and H. Daniel. (1998). Deltaaminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical
implications. J. Clin. Invest. 101: 2761-2767.
32. Eckhardt, U., A. Schroeder, B. Stieger, M. Hochli, L. Landmann, R. Tynes, P. J. Meier, and B. Hagenbuch.
(1999). Polyspecific substrate uptake by the hepatic organic anion transporter Oatp1 in stably transfected CHO
cells. Am. J. Physiol. 276: G1037-G1042.
33. Evers, R., N. H. P. Cnubben, J. Wijnholds, L. van Deemter, P. J. van Bladeren, and P. Borst. (1997). Transport of
glutathione prostaglandin A conjugates by the multidrug resistance protein 1. FEBS Lett. 419: 112-116.
34. 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 MDCK cells expressing cM O AT (MRP2) cDNA. J. Clin. Invest.
101: 1310-1319.
35. Evers, R., G. J. R. Zaman, L. van Deemter, H. Jansen, J. Calafat, L. C. J. M. Oomen, R. P. J. Oude Elferink, P.
Borst, and A. H. Schinkel. (1996). Basolateral localization and export activity of the human multidrug resistanceassociated protein in polarized pig kidney cells. J. Clin. Invest. 97: 1211-1218.
36. Fang, X., F. E. Parkinson, D. A. Mowles, J. D. Young, and C. E. Cass. (1996). Functional characterization of a
recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by
transient expression in cultured mammalian cells. Biochem. J. 317: 457-465.
37. Fei, Y. J., Y. Kanai, S. Nussberger, V. Ganapathy, F. H. Leibach, M. F. Romero, S. K. Singh, W . F. Boron, and M.
A. Hediger. (1994). Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature 368:
563-566.
38. Felipe, A., R. Valdes, B. delSanto, J. Lloberas, J. Casado, and M. PastorAnglada. (1998). Na + -dependent
nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells.
Biochem. J. 330: 997-1001.
39. Flens, M. J., G. J. R. Zaman, P. van der Valk, M. A. Izquierdo, A. B. Schroeiers, G. L. Scheffer, P. van der Groep,
M. de Haas, C. J. L. M. Meijer, and R. J. Scheper. (1996). Tissue distribution of the multidrug resistance protein.
Am. J. Pathol. 148: 1237-1247.
40. Franco, R., J. J. Centelles, and R. K. Kinne. (1990). Further characterization of adenosine transport in renal brushborder membranes. Biochim. Biophys. Acta 1024: 241-248.
41. Friesema, E. C. H., R. Docter, E. P. C. M. Moerings, B. Stieger, B. Hagenbuch, P. J. Meier, E. P. Krenning, G.
Hennemann, and T. J. Visser. (1999). Identification of thyroid hormone transporters. Biochem. Biophys. Res.
Commun. 497-501.
42. Fromm, M. F., B. Leake, D. M. Roden, G. R. Wilkinson, and R. B. Kim. (1999). Human MRP3 transporter:
identification of the 5'-flanking region, genomic organization and alternative splice variants. Biochim. Biophys.
Acta 1415: -374.
43. Ganapathy, M. E., M. Brandsch, P. D. Prasad, V. Ganapathy, and F. H. Leibach. (1995). Differential recognition
of beta -lactam antibiotics by intestinal and renal peptide transporters, PEPT 1 and PEPT 2. J. Biol. Chem. 270:
25672-25677.
44. Ganapathy, M. E., W . Huang, H. Wang, V. Ganapathy, and F. H. Leibach. (1998). Valacyclovir: a substrate for
the intestinal and renal peptide transporters PEPT1 and PEPT2. Biochem. Biophys. Res. Commun. 246: 470-475.
45. Ganapathy, M. E., P. D. Prasad, B. Mackenzie, V. Ganapathy, and F. H. Leibach. (1997). Interaction of anionic
cephalosporins with the intestinal and renal peptide transporters PEPT 1 and PEPT 2. Biochim. Biophys. Acta
1324: 296-308.
46. Gekle, M., S. Mildenberger, C. Sauvant, D. Bednarczyk, S. H. Wright, and W . H. Dantzler. (1999). Inhibition of
initial transport rate of basolateral organic anion carrier in renal PT by BK and phenylephrine. Am. J. Physiol.
277: F251-F256.
47. Gekle, M. and S. Silbernagl. (1994). The role of the proximal tubule in ochratoxin A nephrotoxicity in vivo:
toxodynamic and toxokinetic aspects. Ren. Physiol. Biochem. 17: 40-49.
37
Chapter One
48. Goldberg, J. P. and R. J. Anderson. Renal metabolism and excretion of drugs. In: The kidney: physiology and
pathophysiology, edited by D. W . Seldin and G. Giebisch. New York: Raven Press, Ltd. (1985). p. 2097-2110.
49. Goldstein, E. J. and I. M. Arias. (1976). Interaction of ligandin with radiographic contrast media. Invest. Radiol.
11: 594-597.
50. Grant, C. E., G. Valdimarsson, D. R. Hipfner, K. C. Almquist, S. P. C. Cole, and R. G. Deeley. (1994).
Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs.
Cancer Res. 54: 357-361.
51. Gutierrez, M. M. and K. M. Giacomini. (1993). Substrate selectivity, potential sensitivity and stoichiometry of
Na( +)-nucleoside transport in brush border membrane vesicles from human kidney. Biochim. Biophys. Acta
1149: 202-208.
52. Haase, W., A. Schafer, H. Murer, and R. Kinne. (1978). Studies on the orientation of brush-border membrane
vesicles. Biochem. J. 172: 57-62.
53. Hagenbuch, B., G. Stange, and H. Murer. (1985). Transport of sulphate in rat jejunal and rat proximal tubular
basolateral membrane vesicles. Pflugers Arch. 405: 202-208.
54. Halpin, P. A. and J. L. Renfro. (1996). Renal organic anion secretion: evidence for dopaminergic and adrenergic
regulation. Am. J. Physiol. 271: R1372-9.
55. Han, H., R. L. de Vrueh, J. K. Rhie, K. M. Covitz, P. L. Smith, C. P. Lee, D. M. Oh, W . Sadee, and G. L. Amidon.
(1998). 5'-Amino acid esters of antiviral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1
peptide transporter. Pharm. Res. 15: 1154-1159.
56. Heany, C., H. Shalev, K. Elbedour, R. Carmi, J. B. Staack, V. C. Sheffield, and D. R. Beier. (1998). Human
autosomal recessive osteopetrosis maps to 11 q 13, a position predicted by comparative mapping of the murine
osteosclerosis (oc) mutation. Hum. Mol. Gen. 7: 1407-1414.
57. Hirohashi, T., H. Suzuki, K. Ito, K. Ogawa, K. Kume, T. Shimizu, and Y. Sugiyama. (1998). Hepatic expression of
multidrug resistance-associated
protein-like
proteins maintained
in Eisai
hyperbilirubinemic
rats.
Mol.
Pharmacol. 53: 1068-1075.
58. Hirohashi, T., H. Suzuki, and Y. Sugiyama. (1999). Characterization of the transport properties of cloned rat
multidrug resistance-associated protein 3 (MRP3). J. Biol. Chem. 274: 15181-15185.
59. Hosoyamada, M., T. Sekine, Y. Kanai, and H. Endou. (1999). Molecular cloning and functional expression of a
multispecific organic anion transporter from human kidney. Am. J. Physiol. 276: F122-F128.
60. Huang, Q., S. Yao, M. Ritzel, A. Paterson, C. Cass, and J. Young. (1994). Cloning and functional expression of a
complementary DNA encoding a mammalian nucleoside transport protein. J. Biol. Chem. 17757-17760.
61. Hughey, R. P., B. B. Rankin, J. S. Elce, and N. P. Curthoys. (1978). Specificity of a particulate rat renal peptidase
and its localization along with other enzymes of mercapturic acid synthesis. Arch. Biochem. Biophys. 186: 211
217.
62. Hume, R., M. W . Coughtrie, and B. Burchell. (1995). Differential localisation of UDP-glucuronosyltransferase in
kidney during human embryonic and fetal development. Arch. Toxicol. 69: 242-247.
63. Ishikawa, T., M. Müller, C. Klünemann, T. P. Schaub, and D. Keppler. (1990). ATP-dependent primary active
transport of cysteinyl leukotrienes across liver canalicular membranes. J. Biol. Chem. 265: 19279-19286.
64. Ishizuka, H., K. Konno, H. Naganuma, K. Nishimura, H. Kouzuki, H. Suzuki, B. Stieger, P. J. Meier, and Y.
Sugiyama. (1998). Transport of temocaprilat into rat hepatocytes: Role of organic anion transporting polypeptide.
J. Pharmacol. Exp. Ther. 37-42.
65. Ishizuka, H., K. Konno, H. Naganuma, K. Sasahara, Y. Kawahara, K. Niinuma, H. Suzuki, and Y. Sugiyama.
(1997). Temocaprilat, a novel angiotensin-converting enzyme inhibitor, is excreted in bile via an ATP-dependent
active transporter (cMOAT) that is deficient in Eisai hyperbilirubinemic mutant rats (EHBR). J. Pharmacol. Exp.
Ther. 280: 1304-1311.
66. Ito,
K., H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, and Y. Sugiyama. (1997). Molecular cloning of canalicular
multispecific organic anion transporter defective in EHBR. Am. J. Physiol. 272: G16-G22.
67. 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.
38
68. Jacquemin,
E., B. Hagenbuch, B. Stieger, A. W . Wolkoff, and P. J. Meier. (1994). Expression cloning of a rat liver
Na( +)-independent organic anion transporter. Proc. Natl. Acad. Sci. USA 91: 133-137.
69. Jedlitschky, G., I. Leier, U. Buchholz, K. Barnouin, G. Kurz, and D. Keppler. (1996). Transport of glutathione,
glucuronate and sulfate conjugates by the MRP gene-encoded conjugate export pump. Cancer Res. 56: 988-994.
70. 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.
71. Kanai, N., R. Lu, Y. Bao, A. W . Wolkoff, and V. L. Schuster. (1996). Transient expression of oatp organic anion
transporter in mammalian cells: identification of candidate substrates. Am. J. Physiol. 270: F319-F325.
72. Kanai, N., R. Lu, Y. Bao, A. W . Wolkoff, M. Vore, and V. L. Schuster. (1996). Estradiol 17 beta-D-glucuronide is
a high-affinity substrate for oatp organic anion transporter. Am. J. Physiol. 270: F326-31.
73. Karniski, L. P., M. Lotscher, M. Fucentese, H. Hilfiker, J. Biber, and H. Murer. (1998). Immunolocalization of sat1 sulfate/oxalate/bicarbonate anion exchanger in the rat kidney. Am. J. Physiol. 44: F79-F87.
74. Kartenbeck, J., U. Leuschner, R. Mayer, and D. Keppler. (1996). Absence of the canalicular isoform of the MRP
gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson Syndrome. Hepatology 23: 1061
1066.
75. 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.
76. Keppler, D., J. König, T. P. Schaub, and I. Leier. Molecular basis of ATP-dependent transport of anionic
conjugates in 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.
77. Kirsch, R., G. Fleischner, K. Kamisaka, and I. M. Arias. (1975). Structural and functional studies of ligandin, a
major renal organic anion-binding protein. J. Clin. Invest. 55: 1009-1019.
78. Kiuchi, Y., H. Suzuki, T. Hirohashi, C. A. Tyson, and Y. Sugiyama. (1998). cDN A cloning and inducable
expression of the human multidrug resistance-associated protein 3 (MRP3). FEBS Lett. 433: 149-152.
79. Kobayashi, K., S. Komatsu, T. Nishi, H. Hara, and K. Hayashi. (1991). ATP-dependent transport for glucuronides
in canalicular plasma membrane vesicles. Biochem. Biophys. Res. Com. 176: 622-626.
80. Koepsell, H., V. Gorboulev, and P. Arndt. (1999). Molecular pharmacology of organic cation transporters in
kidney. J. Membrane Biol. 167: 103-117.
81. 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 cDN A enhances drug sensitivity in
human hepatic cancer cells. Cancer Res. 57: 5475-5479.
82. 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 resistanceassociated protein gene (MRP1) in human cancer cell lines. Cancer Res. 57: 3537-3547.
83. Kool, M., M. van der Linden, M. de Haas, F. Baas, and P. Borst. (1999). Expression of human MRP6, a
homologue of the multidrug resistance protein gene MRP1, in tissues and cancer cells. Cancer Res. 59: 175-182.
84. Kool, M., M. van der Linden, M. de Haas, G. L. Scheffer, J. M. L. de Vree, A. J. Smith, G. Jansen, G. J. Peters, N.
Ponne, R. J. Scheper, R. P. J. Oude Elferink, F. Baas, and P. Borst. (1999). MRP3, an organic anion transporter
able to transport anti-cancer drugs. Proc. Natl. Acad. Sci. USA 96: 6914-6919.
85. König, J., D. Rost, Y. Cui, and D. Keppler. (1999). Characterization of the human multidrug resistance protein
isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology 29: 1156-1163.
86.
Kuo, S. M. and P. S. Aronson. (1988). Oxalate transport via the sulfate/HCO3 exchanger in rabbit renal
basolateral membrane vesicles. J. Biol. Chem. 263: 9710-9717.
87. Kusuhara, H., Y. H. Han, M. Shimoda, E. Kokue, H. Suzuki, and Y. Sugiyama. (1998). Reduced folate derivatives
are endogenous substrates for cMOAT in rats. Am. J. Physiol. 275: G789-96.
88.
Kusuhara, H., T. Sekine, N. Utsunomiya-Tata, M. Tsuda, R. kojima, S. H. Cha, Y. Sugiyama, Y. Kanai, and H.
Endou. (1999). Molecular cloning and characterization of a new multispecific organic anion transporter from rat
brain. J. Biol. Chem 274: 13675-13680.
39
Chapter One
89. Kuze, K., P. Graves, A. Leahy, P. Wilson, H. Stuhlmann, and G. You. (1999). Heterologous expression and
functional characterization of a mouse renal organic anion transporter in mammalian cells. J. Biol. Chem. 274:
1519-1524.
90. Lee, K., G. Martin, D. W . Bell, J. R. Testa, and G. D. Kruh. (1998). Isolation of MOAT-B, a widely expressed
multidrug resistance-associated protein/ canalicular multispecific organic anion transporter-related transporter.
Cancer Res. 58: 2741-2747.
91. Leibach, F. H. and V. Ganapathy. (1996). Peptide transporters in the intestine and the kidney. Annu. Rev. Nutr.
16: 99-119.
92. Leier, I., G. Jedlitschky, U. Buchholz, M. Center, S. P. C. Cole, R. G. Deeley, and D. Keppler. (1996). ATPdependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump.
Biochem. J. 314: 433-437.
93. Leier, I., G. Jedlitschky, U. Buchholz, S. P. C. Cole, R. G. Deeley, and D. Keppler. (1994). The MRP gene
encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J. Biol. Chem.
269: 27807-27810.
94. Li, L. Q., T. K. Lee, P. J. Meier, and N. Ballatori. (1998). Identification of glutathione as a driving force and
leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic anion solute transporter. J. Biol. Chem.
273: 16184-16191.
95. Liang, R., Y. J. Fei, P. D. Prasad, S. Ramamoorthy, H. Han, T. L. Yang Feng, M. A. Hediger, V. Ganapathy, and F.
H. Leibach.
(1995).
Human
intestinal
H+/peptide cotransporter.
Cloning,
functional
expression,
and
chromosomal localization. J. Biol. Chem. 270: 6456-6463.
96. Litwack, G., B. Ketterer, and I. M. Arias. (1971). Ligandin: a hepatic protein which binds steroids, bilirubin,
carcinogens and a number of exogenous organic anions. Nature 234: 466-467.
97. Lock, E. A. and C. J. Reed. (1998). Xenobiotic metabolizing enzymes of the kidney. Toxicol. Pathol. 26: 18-25.
98. Loe, D. W., K. C. Almquist, S. P. C. Cole, and R. G. Deeley. (1996). ATP-dependent 17D-estradiol 17-(D-Dglucuronide) transport by multidrug resistance protein (MRP). J. Biol. Chem. 271: 9683-9689.
99. 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.
100. 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: 5130-5136.
101. Loe, D. W., R. K. Stewart, T. E. Massey, R. G. Deeley, and S. P. C. Cole. (1997). ATP-dependent transport of
aflatoxin B 1 and its glutathione conjugates by the product of the multidrug resistance protein (MRP) gene. Mol.
Pharmacol. 51: 1034-1041.
102. Lohr, J. W., G. R. Willsky, and M. A. Acara. (1998). Renal drug metabolism. Pharmacol. Rev. 50: 107-141.
103. Lopez Nieto, C. E., G. You, K. T. Bush, E. J. Barros, D. R. Beier, and S. K. Nigam. (1997). Molecular cloning
and characterization of NKT, a gene product related to the organic cation transporter family that is almost
exclusively expressed in the kidney. J. Biol. Chem. 272: 6471-6478.
104. Lorico, A., G. Rappa, R. A. Finch, D. Yang, R. A. Flavell, and A. C. Sartorelli. (1997). Disruption of the murine
MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels
of glutathione. Cancer Res. 57: 5238-5242.
105. 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.
106. 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.
107. Madon, J., B. Hagenbuch, T. Gerloff, L. Landmann, P. J. Meier, and B. Stieger. (1998). Identification of a novel
multidrug resistance-associated protein (mrp6) at the lateral plasma membrane of rat hepatocytes. Hepatology
28: 400A (abstract).
40
108. 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.
109. Masereeuw, R., F. G. M. Russel, and D. S. Miller. (1996). Multiple pathways of organic anion secretion in renal
proximal tubule revealed by confocal microscopy. Am. J. Physiol. 271: F1173-F1182.
110. Masereeuw, R., W . C. Saleming, D. S. Miller, and F. G. Russel. (1996). Interaction of fluorescein with the
dicarboxylate carrier in rat kidney cortex mitochondria. J. Pharmacol. Exp. Ther. 279: 1559-1565.
111. Masereeuw, R., S. A. Terlouw, R. A. M. H. van Aubel, F. G. M. Russel, and D. S. Miller. (2000). Endothelin B
receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule. Mol. Pharmacol., in press.
112. Masereeuw, R., E. J. van den Bergh, R. J. Bindels, and F. G. Russel. (1994). Characterization of fluorescein
transport in isolated proximal tubular cells of the rat: evidence for mitochondrial accumulation J. Pharmacol.
Exp. Ther. 269: 1261-1267.
113. Masuda, M., Y. I'izuka, M. Yamazaki, R. Nishigaki, Y. Kato, K. Ni'inuma, H. Suzuki, and Y. Sugiyama. (1997).
Methotrexate is excreted into the bile by canalicular multispecific organic anion transporter in rats. Cancer
Res. 57: 3506-3510.
114. Masuda, M., K. Ibaramoto, A. Takeuchi, H. Saito, Y. Hashimoto, and K. I. Inui. (1999). Cloning and functional
characterization of a new multispecific organic anion transporter, OAT-K2, in rat kidney. Mol. Pharmacol. 55:
743-753.
115. Masuda, M., A. Takeuchi, H. Saito, Y. Hashimoto, and K.-I. Inui. (1999). Functional analysis of rat renal organic
anion transporter OAT-K1: bidirectional methotrexate transport in apical membrane. FEBS Lett. 459: 128-132.
116. Masuda, S., H. Saito, and K. I. Inui. (1997). Interactions of nonsteroidal anti-inflammatory drugs with rat renal
organic anion transporter, OAT-K1. J. Pharmacol. Exp. Ther. 283: 1039-1042.
117. 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.
118. McAleer, M. A., M. A. Breen, N. L. White, and N. Matthews. (1999). pABC11 (also known as MOAT-C and
MRP5), a member of the ABC family of proteins, has anion transporter activity but does not confer multidrug
resistance when overexpressed in human embryonic kidney 293 cells. J. Biol. Chem 274: 23541-23548.
119. Meier, P. J., U. Eckhardt, A. Schroeder, B. Hagenbuch, and B. Stieger. (1997). Substrate specificity of sinusoidal
bile acid and organic anion uptake systems in rat and human liver. Hepatology 26: 1667-1677.
120. Merlin, D., A. Steel, A. T. Gewirtz, M. Si Tahar, M. A. Hediger, and J. L. Madara. (1998). hPepT1-mediated
epithelial transport of bacteria-derived chemotactic peptides enhances neutrophil-epithelial interactions. J.
Clin. Invest. 102: 2011-2018.
121. Miller, D. S. (1998). Protein kinase C regulation of organic anion transport in renal proximal tubule. Am. J.
Physiol. 274: F156-64.
122. Miller, D. S., D. M. Barnes, and J. B. Pritchard. (1994). Confocal microscopic analysis of fluorescein
compartmentation within crab urinary bladder cells. Am. J. Physiol. 267: R16-25.
123. Miller, D. S., S. Letcher, and D. M. Barnes. (1996). Fluorescence imaging study of organic anion transport from
renal proximal tubule cell to lumen. Am. J. Physiol. 271: F508-F520.
124. Miller, D. S. and J. B. Pritchard. (1994). Nocodazole inhibition of organic anion secretion in teleost renal
proximal tubules. Am. J. Physiol. 267: R695-704.
125. Miller, D. S., D. E. Stewart, and J. B. Pritchard. (1993). Intracellular compartmentation of organic anions within
renal cells. Am. J. Physiol. 264: R882-90.
126. Miyamoto, K., T. Shiraga, K. Morita, H. Yamamoto, H. Haga, Y. Taketani, I. Tamai, Y. Sai, A. Tsuji, and E.
Takeda. (1996). Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide
transporter. Biochim. Biophys. Acta 1305: 34-38.
127. Miyamoto, Y., J. L. Coone, V. Ganapathy, and F. H. Leibach. (1988). Distribution and properties of the
glycylsarcosine-transport system in rabbit renal proximal tubule. Studies with isolated brush-border-membrane
41
Chapter One
128. Miyamoto, Y., V. Ganapathy, and F. H. Leibach. (1985). Proton gradient-coupled uphill transport of
glycylsarcosine in rabbit renal brush-border membrane vesicles. Biochem. Biophys. Res. Commun. 132: 946
953.
129. Monks, T. J. and S. S. Lau. (1987). Renal transport processes and glutathione conjugate-mediated
nephrotoxicity. Drug Metab. Dispos. 15: 437-441.
130. Muraoka, I., T. Hasegawa, M. Nadai, L. Wang, S. Haghgoo, O. Tagaya, and T. Nabeshima. (1995). Biliary and
renal excretions of cefpiramide in Eisai hyperbilirubinemic rats. Antimicrob. Agents Chemother. 39: 70-74.
131. Murata, M., I. Tamai, Y. Sai, O. Nagata, H. Kato, Y. Sugiyama, and A. Tsuji. (1998). Hepatobiliary transport
kinetics of HSR-903, a new quinolone antibacterial agent. Drug Metab. Dispos. 26: 1113-1119.
132. Müller, M., C. J. L. M. Meijer, P. Borst, R. J. Scheper, N. H. Mulder, E. G. de Vries, and P. L. M. Jansen. (1994).
Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATPdependent glutathione S-conjugate transport. Proc. Natl. Acad. Sci. USA 91: 13033-13037.
133. Newton, J. F., D. A. Pasino, and J. B. Hook. (1985). Acetaminophen nephrotoxicity in the rat: quantitation of
renal metabolic activation in vivo. Toxicol. Appl. Pharmacol. 78: 39-46.
134. Nies, A. T., T. Cantz, M. Brom, I. Leier, and D. Keppler. (1998). Expression of the apical conjugate export
pump, Mrp2, in the polarized hepatoma cell line, WIF-B. Hepatology 28: 1332-1340.
135. Niinuma, K., O. Takenaka, T. Horie, K. Kobayashi, Y. Kato, H. Suzuki, and Y. Sugiyama. (1997). Kinetic
analysis of the primary active transport of conjugated metabolites across the bile canalicular membrane:
Comparative
study
of
S-(2,4-dinitrophenyl)-glutathione
and
6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-
pyridylmethyl) benzothiazole glucuronide. J. Pharmacol. Exp. Ther. 866-872.
136. Oude Elferink, R. P. J. and P. L. M. Jansen. (1994). The role of the canalicular multispecific organic anion
transporter in the disposal of endo- and xenobiotics. Pharmacol. Ther. 64: 77-97.
137. Pajor, A. M. (1998). Sequence of a pyrimidine-selective Na+/nucleoside cotransporter from pig kidney,
pkCNT1. Biochim. Biophys. Acta 1415: 266-269.
138. Pang, K. S., P. J. Wang, A. Y. K. Chung, and A. W . Wolkoff. (1998). The modified dipeptide, enalapril, an
angiotensin-converting enzyme inhibitor, is transported by the rat liver organic anion transport protein.
Hepatology 28: 1341-1346.
139. Pastor-Anglada, M., A. Felipe, and F. Javier Casado. (1998). Transport and mode of action of nucleoside
derivatives used in chemical and antiviral therapies. Trends Pharmacol. Sci. 19: 424-430.
140. 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 resistanceassociated protein gene. Science 271: 1126-1128.
141. 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.
142. 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.
143. Pegg, D. G. and J. B. Hook. (1977). Glutathione S-transferases: an evaluation of their role in renal organic anion
transport. J. Pharmacol. Exp. Ther. 200: 65-74.
144. 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.
145. Priebe, W ., M. Krawczyk, M. T. Kuo, Y. Yamane, N. Savaraj, and T. Ishikawa. (1998). Doxorubicin- and
daunorubicin-glutathione conjugates, but not unconjugated drugs, competitively inhibit leukotriene C-4
transport mediated by MRP/GS-X pump. Biochem. Biophys. Res. Com. 247: 859-863.
146. Pritchard, J. B. (1995). Intracellular alpha-ketoglutarate controls the efficacy of renal organic anion transport. J.
Pharmacol. Exp. Ther. 274: 1278-1284.
147. Pritchard, J. B. and D. S. Miller. (1993). Mechanisms mediating renal secretion of organic anions and cations.
Physiol. Rev. 73: 765-796.
42
148. Pritchard, J. B. and J. L. Renfro. (1983). Renal sulfate transport at the basolateral membrane is mediated by
anion exchange. Proc. Natl. Acad. Sci. USA 80: 2603-2607.
149. Pulaski, L., G. Jedlitschky, I. Leier, U. Buchholz, and D. Keppler. (1996). Identification of the multidrugresistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes. Eur. J. Biochem. 241:
644-648.
150. Race, J. E., S. M. Grassl, W . J. Williams, and E. J. Holtzman. (1999). Molecular cloning and characterization of
two novel human renal organic anion transporters (hOAT1 and hOAT3). Biochem. Biophys. Res. Com. 255:
508-514.
151. Ramamoorthy, S., W . Liu, Y. Y. Ma, T. L. Yang Feng, V. Ganapathy, and F. H. Leibach. (1995). Proton/peptide
cotransporter (PEPT 2) from human kidney: functional characterization and chromosomal localization.
Biochim. Biophys. Acta 1240: 1-4.
152. Reid, G., N. A. Wolff, F. M. Dautzenberg, and G. Burckhardt. (1998). Cloning of a human renal paminohippurate transporter, hROAT1. Kidney Blood Press. Res. 21: 233-237.
153. Ritzel, M. W ., S. Y. Yao, M. Y. Huang, J. F. Elliott, C. E. Cass, and J. D. Young. (1997). Molecular cloning and
functional expression of cDNAs encoding a human Na + -nucleoside cotransporter (hCNT1). Am. J. Physiol.
272: C707-14.
154. Ritzel, M. W . L., S. Y. M. Yao, A. M. L. Ng, J. R. Mackey, C. E. Cass, and J. D. Young. (1998). Molecular
cloning, functional expression and chromosomal localization of a cDNA encoding a human Na +/nucleoside
cotransporter (hCNT2) selective for purine nucleosides and uridine. Mol. Membr. Biol. 15: 203-211.
155. Roch-Ramel, F. (1998). Renal transport of organic anions. Curr. Opin. Nephrol. Hypertens. 7: 517-524.
156. Roelofsen, H., R. Ottenhoff, R. P. J. Oude Elferink, and P. L. M. Jansen. (1991). Hepatocanalicular organicanion transport is regulated by protein kinase C. Biochem. J. 278: 637-641.
157. Roelofsen, H., C. J. Soroka, D. Keppler, and J. L. Boyer. (1998). Cyclic AMP stimulates sorting of the canalicular
organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets. J. Cell Sci. 111: 1137
1145.
158. 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.
159. Saito, H., M. Okuda, T. Terada, S. Sasaki, and K. Inui. (1995). Cloning and characterization of a rat H+/peptide
cotransporter mediating absorption of beta-lactam antibiotics in the intestine and kidney. J. Pharmacol. Exp.
Ther. 275: 1631-1637.
160. Saito, H., T. Terada, M. Okuda, S. Sasaki, and K. Inui. (1996). Molecular cloning and tissue distribution of rat
peptide transporter PEPT2. Biochim. Biophys. Acta 1280: 173-177.
161. Satlin, L. M., V. Amin, and A. W . Wolkoff. (1997). Organic anion transporting polypeptide mediates organic
anion/ HCO3- exchange. J. Biol. Chem. 272: 26340-26345.
162. Schaner, M. E., J. Wang, S. Zevin, K. M. Gerstin, and K. M. Giacomini. (1997). Transient expression of a
purine-selective nucleoside transporter ( SPNTint) in a human cell line (HeLa). Pharm. Res. 14: 1316-1321.
163. 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.
164. Schuetz, J. D., M. C. Connelly, D. Sun, S. G. Paibir, P. M. Flynn, R. V. Srinivas, A. Kumar, and A. Fridland.
(1999). MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs. Nature Med.
5: 1048-1051.
165. Sekine, T., S. H. Cha, M. Tsuda, N. Apiwattanakul, N. Nakajima, Y. Kanai, and H. Endou. (1998). Identification
of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett. 429: 179-182.
166. 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.
167. Sheehan, D. and T. J. Mantle. (1984). Evidence for two forms of ligandin (YaYa dimers of glutathione Stransferase) in rat liver and kidney. Biochem. J. 218: 893-897.
168. Sheikh Hamad, D., K. Timmins, and Z. Jalali. (1997). Cisplatin-induced renal toxicity: possible reversal by Nacetylcysteine treatment. J. Am. Soc. Nephrol. 8: 1640-1644.
43
Chapter One
169. Shen, H., D. E. Smith, T. Yang, Y. G. Huang, J. B. Schnermann, and F. C. Brosius III. (1999). Localization of
PEPT1 and PEPT2 proton-coupled oligopeptide transporter mRNA and protein in rat kidney. Am. J. Physiol.
276: F658-F665.
170. Siess, E. A., D. G. Brocks, and O. H. Wieland. (1976). Subcellular distribution of key metabolites in isolated
liver cells from fasted rats. FEBS Lett. 69: 265-271.
171. Silbernagl, S., V. Ganapathy, and F. H. Leibach. (1987). H+ gradient-driven dipeptide reabsorption in proximal
tubule of rat kidney. Studies in vivo and in vitro. Am. J. Physiol. 253: F448-57.
172. Simonson, G. D., A. C. Vincent, K. J. Roberg, Y. Huang, and V. Iwanij. (1994). Molecular cloning and
characterization of a novel liver-specific transport protein. J. Cell Sci. 107: 1065-1072.
173. Soboll, S., R. Scholz, M. Friesl, R. Elbers, and H. W . Heldt. Distribution of metabolites between mitochondria
and cytosol of perfused liver. In: Use of isolated liver cells and kidney tubules in metabolic studies, edited by J.
M. Tager, H. D. Söling, and J. R. Williamson. Amsterdam: North-Holland Publishing Company, (1975). p. 29
40.
174. Stride, B. D., C. E. Grant, D. W . Loe, D. R. Hipfner, S. P. C. Cole, and R. G. Deeley. (1997). Pharmacological
characterization of the murine and human orthologs of multidrug-resistance protein in transfected human
embryonic kidney cells. Mol. Pharmacol. 52: 344-353.
175. Suzuki, H. and Y. Sugiyama. (1998). Excretion of GSSG and glutathione conjugates mediated by MRP1 and
cMOAT/MRP2. Semin. Liver Dis. 18: 359-376.
176. 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.
177. Takahashi, K., N. Nakamura, T. Terada, T. Okano, T. Futami, H. Saito, and K. I. Inui. (1998). Interaction of
beta-lactam antibiotics with H+/peptide cotransporters in rat renal brush-border membranes. J. Pharmacol.
Exp. Ther. 286: 1037-1042.
178. Takano, M., J. Nagai, M. Yasuhara, and K. Inui. (1996). Regulation of p-aminohippurate transport by protein
kinase C in OK kidney epithelial cells. Am. J. Physiol. 271: F469-75.
179. 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.
180. Tamai, I., T. Nakanishi, H. Nakahara, Y. Sai, V. Ganapathy, F. H. Leibach, and A. Tsuji. (1998). Improvement
of L-dopa absorption by dipeptidyl derivation, utilizing peptide transporter PepTi. J. Pharm. Sci. 87: 1542
1546.
181. 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.
182. Temple, C. S. and C. A. Boyd. (1998). Proton-coupled oligopeptide transport by rat renal cortical brush border
membrane vesicles: a functional analysis using ACE inhibitors to determine the isoform of the transporter.
183. Terada, T., H. Saito, M. Mukai, and K. Inui. (1997). Recognition of beta-lactam antibiotics by rat peptide
transporters, PEPT1 and PEPT2, in LLC-PK1 cells. Am. J. Physiol. 273: F706-11.
184. Terlouw, S. A., F. G. M. Russel, and R. Masereeuw. (1999). Metabolite anion carriers mediate the uptake of the
anionic drug fluorescein in renal cortical mitochondria. Fund. Clin. Pharmacol. 13: 140 (abstract).
185. Tiruppathi, C., V. Ganapathy, and F. H. Leibach. (1990). Kinetic evidence for a common transporter for
glycylsarcosine and phenylalanylprolylalanine in renal brush-border membrane vesicles. J. Biol. Chem. 265:
14870-14874.
186. Tojo, A., T. Sekine,
N.
Nakajima, M.
Hosoyamada, Y. Kanai, K. Kimura, and H. Endou. (1999).
Immunohistochemical localization of multispecific renal organic anion transporter 1 in rat kidney. J. Am. Soc.
Nephrol. 10: 464-471.
187. Tsuda, M., T. Sekine, M. Takeda, S. H. Cha, Y. Kanai, M. Kimura, and H. Endou. (1999). Transport of
ochratoxin A by renal multispecific organic anion transporter 1. J. Pharmacol. Exp. Ther. 289: 1301-1305.
44
188. Uchiumi, T., E. Hinoshita, S. Haga, T. Nakamura, T. Tanaka, S. Toh, T. Furukawa, T. Kawabe, M. Wada, K.
Kagotani, K. Okumura, K. Kohno, S.-I. Akiyama, and M. Kuwano. (1998). Isolation of a novel human
canalicular multispecific organic anion transporter, cMOAT2/MRP3, and its expression in cisplatin-resistant
cancer cells with decreased ATP-dependent drug transport. Biochem. Biophys. Res. Com. 252: 103-110.
189. Ullrich, K. J. (1997). Renal transporters for organic anions and cations. Structural requirements for substrates. J.
Membrane Biol. 158: 95-107.
190. Ullrich, K. J. and G. Rumrich. (1997). Luminal transport step of para-aminohippurate (PAH): transport from
PAH-loaded proximal tubular cells into the tubular lumen of the rat kidney in vivo. Pflugers Arch. 433: 735
743.
191. 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.
192. van Aubel, R. A. M. H., J. B. Koenderink, J. G. P. Peters, C. H. van Os, and F. G. M. Russel. (1999).
Mechanisms 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.
193. van Aubel, R. A. M. H., J. G. P. Peters, R. Masereeuw, C. H. van Os, and F. G. M. Russel. (1999). The
multidrug resistance protein Mrp2
mediates ATP-dependent transport of the classical organic anion
p-aminohippurate. Submitted
194. 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 resistanceassociated protein Mrp2 expressed in insect cells. Mol. Pharmacol. 53: 1062-1067.
195. Versantvoort, C. H., H. J. Broxterman, T. Bagrij, R. J. Scheper, and P. R. Twentyman. (1995). Regulation by
glutathione of drug transport in multidrug- resistant human lung tumour cell lines overexpressing multidrug
resistance- associated protein. Br. J. Cancer 72: 82-89.
196. Wada, M., S. Toh, K. Taniguchi, T. Nakamura, T. Uchiumi, K. Kohno, I. Yoshida, A. Kimura, S. Sakisaka, Y.
Adachi, and M. Kuwano. (1998). Mutations in the canalicular multispecific organic anion transporter (cMOAT)
gene, a novel ABC transporter, in patients with hyperbilirubinemia II/ Dubin-Johnson syndrome. Hum. Mol.
Gen. 7: 203-207.
197. Wang, H., Y. J. Fei, V. Ganapathy, and F. H. Leibach. (1998). Electrophysiological characteristics of the proton
coupled peptide transporter PEPT2 cloned from rat brain. Am. J. Physiol. 275: C967-75.
198. Wang, J., S. F. Su, M. J. Dresser, M. E. Schaner, C. B. Washington, and K. Giacomini. (1997). Na( + )-dependent
purine nucleoside transporter from human kidney: cloning and functional characterization. Am. J. Physiol.
273: F1058-65.
199. Welborn, J. R., S. Shpun, W . H. Dantzler, and S. H. Wright. (1998). Effect of alpha-ketoglutarate on organic
anion transport in single rabbit renal proximal tubules. Am. J. Physiol. 274: F165-74.
200. Wenzel, U., I. Gebert, H. Weintraut, W . M. Weber, W . Clauss, and H. Daniel. (1996). Transport characteristics
of differently charged cephalosporin antibiotics in oocytes expressing the cloned intestinal peptide transporter
PepT1 and in human intestinal Caco-2 cells. J. Pharmacol. Exp. Ther. 277: 831-839.
201. 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.
202. Wijnholds, J., C. A. Mol, G. L. Scheffer, R. J. Scheper, and P. Borst. (1999). Multidrug resistance protein 5, a
candidate multispecific organic anion transporter. Proc. Amer. Assoc. Cancer Res. 40: 315 (abstract).
203. 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 druginduced damage. J. Exp. Med. 188: 797-808.
204. Williams, T. C. and S. M. Jarvis. (1991). Multiple sodium-dependent nucleoside transport systems in bovine
renal brush-border membrane vesicles. Biochem. J. 274: 27-33.
205. Wolff, N. A., A. Werner, S. Burkhardt, and G. Burckhardt. (1997). Expression cloning and characterization of a
renal organic anion transporter from winter flounder. FEBS Lett. 417: 287-291.
45
Chapter One
206. Yamazaki, M., S. Akiyama, K. Ni'inuma, R. Nishigaki, and Y. Sugiyama. (1997). Biliary excretion of pravastatin
in rats: contribution of the excretion pathway mediated by canalicular multispecific organic anion transporter.
Drug Metab. Dispos. 25: 1123-1129.
207. Yao, S. Y., C. E. Cass, and J. D. Young. (1996). Transport of the antiviral nucleoside analogs 3'-azido-3'deoxythymidine and 2',3'-dideoxycytidine by a recombinant nucleoside transporter (rCNT) expressed in
Xenopus laevis oocytes. Mol. Pharmacol. 50: 388-393.
208. Yao, S. Y., A. M. Ng, M. W . Ritzel, W . P. Gati, C. E. Cass, and J. D. Young. (1996). Transport of adenosine by
recombinant purine- and pyrimidine-selective sodium/nucleoside cotransporters from rat jejunum expressed in
Xenopus laevis oocytes. Mol. Pharmacol. 50: 1529-1535.
209. Yokota, H., H. Inoue, H. Taniyama, T. Kobayashi, H. Iwano, Y. Kagawa, H. Okada, and A. Yuasa. (1997). High
induction of phenol UDP-glucuronosyltransferase in the kidney medulla of beta-naphthoflavone-treated rats.
210. Zaman, G. J. R., N. H. P. Cnubben, P. J. van Bladeren, R. Evers, and P. Borst. (1996). Transport of the
glutathione conjugate of ethacrynic acid by the human multidrug resistance protein MRP. FEBS Lett. 391: 126
130.
211. Zaman, G. J. R., M. J. Flens, M. R. van Leusden, M. de Haas, H. S. Mulder, J. Lankelma, H. M. Pinedo, R. J.
Scheper, F. Baas, H. J. Broxterman, and P. Borst. (1994). The human multidrug resistance-associated protein
MRP is a plasma membrane drug-efflux pump. Proc. Natl. Acad. Sci. USA 91: 8822-8826.
212. 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.
213. Zaman, G. J. R., C. H. Versantvoort, J. J. Smit, E. W . Eijdems, M. de Haas, A. J. Smith, H. J. Broxterman, N. H.
Mulder, E. G. de Vries, F. Baas, and P. Borst. (1993). Analysis of the expression of MRP, the gene for a new
putative transmembrane drug transporter, in human multidrug resistant lung cancer cell lines. Cancer Res. 53:
1747-1750.
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 f 1Pharmacology 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. W e 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 ([3 H]E2 i 7 ßG, 50 nM) and [3 H]leukotriene C4 (3
nM) were taken up by Sf9-Mrp2 membrane vesicles in an ATP-dependent fashion.
Uptake of [3 H]E2 i 7 ßG was dependent on the osmolarity of the medium and
saturable for ATP (Km = 623 ^M). Leukotriene C 4, 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-ßD-glucuronide did not inhibit transport. In conclusion, we show direct ATPdependent transport by recombinant rabbit Mrp2 and provide new data on Mrp2
inhibitor specificity.
(available online at http://molpharm.aspetjournals.org)
49
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 ATPdependent 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 . Ford
Hutchinson (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 (FQ KRQ Q KKSQ KNSRLQ G L) 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 cD N A 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 2690
5407) 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
contains
the
(5'-A TG CTGGATAAGTTCTGCAAC-3';
ATG
start
codon
(underlined),
and
nucleotides 347-368), which
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 cD N A 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 ~ 6 5 % 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).
53
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 anti
rabbit 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, pH
7.4). 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
A
B
200 —
200
KIDNEY
_
+
***4
116 —
Sf9-Mrp2
_
+ PNGase F
—m
116—
97 —
97—
66
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.
1
4
Fig. 2. Comparison of Mrp2 protein
levels in Sf9 cells, liver and renal
Sf9-Mrp2
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
5
20
separated on a 7.5% SDS
polyacrylamide gel and subjected to
immunoblot analysis using antiserum
liver
k78mrp2. Proteins were visualized
using enhanced chemiluminescence.
renal
proximal
tubules
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
tubular cells to immunoblot analysis using antiserum k78mrp2 (Fig. 2). A 1 j g
protein-equivalent of crude membranes from Sf9-Mrp2 cells was sufficient to
detect Mrp2. Approximately 20 j g 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 j g 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 ~11fold 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 ATPdependent [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 (60
8000 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 Kmvalue 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]. W e
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 1
Effect of various compounds on Mrp2-mediated [3H]E217ßG transport
Compound
Concentration
[3H]E2 17ßG
uptake
j
M
% control
100
None (control)
LTC4
10
22 ± 7b
a-Naphthyl-ß-D-glucuronide
1000
94 ± 1
p-Nitrophenyl-ß-D-glucuronide
1000
97 ± 5
Phenolphthalein glucuronide
1000
5 ± 4b
5
33 ± 3a
10
48 ± 1a
MK571
Cyclosporin A (CsA)
Fluorescein (FL)
1000
94 ± 4
Methotrexate (MTX)
1000
55 ± 4a
100
22 ± 7b
Fluorescein-methotrexate (FL-MTX)
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 ATPdependent 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 ATPdependent 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 FLMTX uptake but completely abolished luminal excretion. This suggests that FLMTX 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 E2 I 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 Mrp2substrates, with emphasis on compounds that are excreted by the kidney.
62
Acknowledgments
W e thank A. Hartog for isolation of cells of rabbit renal proximal tubules. W e 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.
References
1.
Bakos, E., T. Hegedüs, Z. Hollo, E. Welker, G.E. Tusnady, G.J.R. Zaman, M.J. Flens, A. Varadi, and B. Sarkadi.
(1996). Membrane topology and glycosylation of the human multidrug resistance-associated protein. ). Biol.
Chem. 271: 12322-12326.
2.
Bergwerk, A.J., X. Shi, A.C. Ford, N. Kanai, E. Jacquemin, R.D. Burk, S. Bai, P.M. Novikoff, B. Stieger, P.J.
Meier, V.L. Schuster, and A.W. Wolkoff. (1996). Immunologic distribution of an organic anion transport
protein in rat liver and kidney. Am. J. Physiol. 271: G231-G238.
3.
Büchler, M., J. König, M. Brom, J. Kartenbeck, H. Spring, T. Horie, and D. Keppler. (1996). cDN A 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.
4.
de Vries, M.H., F.A. Redegeld, A.S. Koster, J. Noordhoek, J.G. de Haan, R.P.J. Oude Elferink, and P.L.M.
Jansen. (1989). Hepatic, intestinal and renal transport of 1-naphthol-beta-D- glucuronide in mutant rats with
5.
Deen, P.M.T., R.A.M.H. van Aubel, A.F. van Lieburg, and C.H. van Os. (1996). Urinary content of aquaporin 1
and 2 in nephrogenic diabetes insipidus. J. Am. Soc. Nephrol. 7: 836-841.
6.
Doige, C.A. and F.J. Sharom. (1991). Strategies for the purification of P-glycoprotein from multidrug-resistant
chinese hamster ovary cells. Protein Expr. Purif. 2: 256-265.
7.
Flens, M.J., G.J.R. Zaman, P. van der Valk, M.A. Izquierdo, A.B. Schroeiers, G.L. Scheffer, P. van der Groep,
M. de Haas, C.J.L.M. Meijer, and R.J. Scheper. (1996). Tissue distribution of the multidrug resistance protein.
Am. J. Pathol. 148: 1237-1247.
8.
Gao, M., D.W. Loe, C.E. Grant, S.P.C. Cole, and R.G. Deeley. (1996). Reconstitution of ATP-dependent
leukotriene C4 transport by co-expression of both half-molecules of human multidrug resistance protein in
insect cells. J. Biol. Chem. 271: 27782-27787.
9.
Haase, W., A. Schafer, H. Murer, and R. Kinne. (1978). Studies on the orientation of brush-border membrane
10.
Huber, M., A. Guhlmann, P.L.M. Jansen, and D. Keppler. (1987). Hereditary defect of hepatobiliary cysteinyl
11.
Ito, K., H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, and Y. Sugiyama. (1997). Molecular cloning of
leukotriene elimination in mutant rats with defective hepatic anion excretion. Hepatology 7: 224-228.
canalicular multispecific organic anion transporter defective in EHBR. Am. J. Physiol. 272: G16-G22.
12.
Ito, K., H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, and Y. Sugiyama. (1998). Functional analysis of a
13.
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.
63
Chapter Two
14.
Kanai, N., R. Lu, Y. Bao, A.W. Wolkoff, M. Vore, and V.L. Schuster. (1996). Estradiol 17 beta-D-glucuronide is
a high-affinity substrate for oatp organic anion transporter. Am. J. Physiol. 270: F326-31.
15.
Kartner, N., J.W . Hanrahan, T.J. Jensen, A.L. Naismith, S.Z. Sun, C.A. Ackerley, E.F. Reyes, L.C. Tsui, J.M.
Rommens, and C.E. Bear. (1991). Expression of the cystic fibrosis gene in non-epithelial invertebrate cells
produces a regulated anion conductance. Cell 64: 681-691.
16.
Klaassen, C.H.W., T.J.F. van Uem, M.P. de Moel, G.L.J. de Caluwe, H.G.P. Swarts, and J.J.H.H.M. de Pont.
(1993). Functional expression of gastric H,K-ATPase using the baculovirus expression system. FEBS Lett. 329:
277-282.
17.
Kobayashi, K., S. Komatsu, T. Nishi, H. Hara, and K. Hayashi. (1991). ATP-dependent transport for
glucuronides in canalicular plasma membrane vesicles. Biochem. Biophys. Res. Com. 176: 622-626.
18.
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 resistanceassociated protein gene (MRP1) in human cancer cell lines. Cancer Res. 57: 3537-3547.
19.
Leier, I., G. Jedlitschky, U. Buchholz, S.P.C. Cole, R.G. Deeley, and D. Keppler. (1994). The MRP gene
encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J. Biol. Chem.
269: 27807-27810.
20.
Li, L.Q., T.K. Lee, P.J. Meier, and N. Ballatori. (1998). Identification of glutathione as a driving force and
leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic anion solute transporter. J. Biol. Chem.
273: 16184-16191.
21.
Loe, D.W., R.G. Deeley, and S.P.C. Cole. (1996). Biology of the multidrug resistance-associated protein, MRP.
Eur. J. Cancer 32A: 945-957.
22.
Madon, J., U. Eckhardt, T. Gerloff, B. Stieger, and P.J. Meier. (1997). Functional expression of the rat liver
23.
Marples, D., M.A. Knepper, E.I. Christensen, and S. Nielsen. (1995). Redistribution of aquaporin-2 water
channels induced by vasopressin in rat inner medullary collecting duct. Am. J. Physiol. 269: C655-C664.
24.
Masereeuw, R., F.G.M. Russel, and D.S. Miller. (1996). Multiple pathways of organic anion secretion in renal
25.
Masuda, S., H. Saito, H. Nonoguchi, K. Tomita, and K.-I. Inui. (1997). mRNA distribution and membrane
26.
Müller, M. and P.L.M. Jansen. (1997). Molecular aspects of hepatobiliary transport. Am. J. Physiol. 272:
localization of the OAT-K1 organic anion transporter in rat renal tubules. FEBS Lett. 407: 127-131
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.
33.
Schaub, T.P., J. Kartenbeck, J. König, O. Vogel, R. Witzgall, W . Kriz, and D. Keppler. (1997). Expression of the
Natl. Acad. Sci. USA 74: 5463-5467.
conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubules. J. Am.
Soc. Nephrol. 8: 1213-1221.
64
34.
Sullivan, L.P., J.A. Grantham, L. Rome, D. Wallace, and J.J. Grantham. (1990). Fluorescein transport in isolated
35.
Takikawa, H., R. Yamazaki, N. Sano, and M. Yamanaka. (1996). Biliary excretion of estradiol-17 beta
36.
Taniguchi, K., M. Wada, K. Kohno, T. Nakamura, T. Kawabe, M. Kawakami, K. Kagotani, K. Okumura, S.-I.
proximal tubules in vitro: epifluorometric analysis. Am. J. Physiol. 258: F46-51.
glucuronide in the rat. Hepatology 23: 607-613.
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 17-(b-D-glucuronide) in
40.
Weinman, S.A. and M .W . Carruth. (1997). MRP-2/cMOAT mediates leukotriene-dependent chloride channel
rat canalicular membrane vesicles. Am. J. Physiol. 271: G791-G798.
activation in hepatocytes. Hepatology 26: 131A (abstract).
65
Chapter 3
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,
Adapted version submitted for publication
67
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.
69
Chapter Three
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 C 4, LTC4; estradiol175-D-glucuronide, E2 I 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].
W e 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.
70
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, pH
7.4). 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 at
100,000 x g for 40 min and the pellet obtained was resuspended in TS-buffer with a tightfitting 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.
71
Chapter Three
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 Trisbuffered 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 anti
rabbit 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 N 2 . 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 M O, USA),
dehydrated by 100%
methanol, mounted in mowiol (Hoechst), and analyzed by
immunofluorescence microscopy.
72
A
*
1200 kDa
g
116 kDa
m
Mrp2
Fig.
mock
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.
membranes
from
liver,
kidney,
and
small
PNGase
F treatment of crude
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.
74
E C H Li Lu K S
Mrp2
A
ß-actin
K
Li
+
B
* •«
•
1
PNGase F
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.
75
Chapter Three
Fig. 3. Immunolocalization of Mrp2 in rabbit small intestine. PLP-fixed sections of rabbit small
intestine
were
incubated
immunofluorescence was
with
polyclonal
antibody
located to the apical
GP8.
A
and
B,
Mrp2-specific
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.
76
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.
77
Chapter Three
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.
78
Discussion
W e 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 basal
lateral 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
79
Chapter Three
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 [m drla (-/-)], 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
80
transport of at least the latter compound [8], Mrp2 might also be a determinant in
the low oral bioavailability of certain (antiviral) drugs.
References
1.
Ambudkar, S.V., S. Dey, C.A. Hrycyna, M. Ramachandra, I. Pastan, and M.M. Gottesman. (1999).
Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu. Rev. Pharmacol.
Toxicol. 39: 361-398.
2.
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.
3.
Chen, X., H. Tsukaguchi, X.-Z. Chen, U.V. Berger, and M.A. Hediger. (1999). Molecular and functional
analysis of SDCT2, a novel rat sodium-dependent dicarboxylate transporter. J. Clin. Invest. 103: 1159-1168.
4.
Cui, Y., J. König, U. Buchholz, H. Spring, I. Leier, and D. Keppler. (1999). Drug resistance and ATPdependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently
expressed in human and canine cells. Mol. Pharmacol. 55: 929-937.
5.
de Vries, M.H., F.A. Redegeld, A.S. Koster, J. Noordhoek, J.G. de Haan, R.P.J. Oude Elferink, and P.L.M.
Jansen. (1989). Hepatic, intestinal and renal transport of 1-naphthol-beta-D- glucuronide in mutant rats with
6.
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 MDCK cells expressing cM O AT (MRP2) cDNA. J.
Clin. Invest. 101: 1310-1319.
7.
Gottesman, M.M. and I. Pastan. (1993). Biochemistry of multidrug resistance mediated by the multidrug
transporter. Annu. Rev. Biochem. 62: 385-427.
8.
Gutmann, H., G. Fricker, J. Drewe, M. Toeroek, and D.S. Miller. (1999). Interactions of HIV protease
9.
Hooijberg, J.H., H.J. Broxterman, M. Kool, Y.G. Assaraf, G.J. Peters, P. Noordhuis, R.J. Scheper, P. Borst,
inhibitors with ATP-dependent drug export proteins. Mol. Pharmacol. 56: 383-389.
H.M. Pinedo, and G. Jansen. (1999). Antifolate resistance mediated by the multidrug resistance proteins
MRP1 and MRP2. Cancer Res. 59: 2532-2535.
10.
Ito, K., H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, and Y. Sugiyama. (1997). Molecular cloning of
canalicular multispecific organic anion transporter defective in EHBR. Am. J. Physiol. 272: G16-G22.
11.
Ito, K., H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, and Y. Sugiyama. (1998). Functional analysis of a
12.
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.
13.
Kiuchi, Y., H. Suzuki, T. Hirohashi, C.A. Tyson, and Y. Sugiyama. (1998). cDN A cloning and inducable
expression of the human multidrug resistance-associated protein 3 (MRP3). FEBS Lett. 433: 149-152.
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 cDN A 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 OAT (MRP2), MRP3, MRP4, and MRP5, homologous of the multidrug
resistance-associated protein gene (MRP1) in human cancer cell lines. Cancer Res. 57: 3537-3547.
81
Chapter Three
16.
Kool, M., M. van der Linden, M. de Haas, F. Baas, and P. Borst. (1999). Expression of human MRP6, a
homologue of the multidrug resistance protein gene MRP1, in tissues and cancer cells. Cancer Res. 59: 175
182.
17.
König, J., D. Rost, Y. Cui, and D. Keppler. (1999). Characterization of the human multidrug resistance protein
18.
Kusuhara, H., Y.H. Han, M. Shimoda, E. Kokue, H. Suzuki, and Y. Sugiyama. (1998). Reduced folate
isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology 29: 1156-1163.
derivatives are endogenous substrates for cM O AT in rats. Am. J. Physiol. 275: G789-96.
19.
Lee, C.G., M.M. Gottesman, C.O. Cardarelli, M. Ramachandra, K.T. Jeang, S.V. Ambudkar, I. Pastan, and S.
Dey. (1998). HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. Biochemistry 37:
3594-3601.
20.
Madon, J., B. Hagenbuch, T. Gerloff, L. Landmann, P.J. Meier, and B. Stieger. (1998). Identification of a
novel multidrug resistance-associated protein (mrp6) at the lateral plasma membrane of rat hepatocytes
(abstract). Hepatology 28: 400A.
21.
Masereeuw, R., M.M. Moons, B.H. Toomey, F.G.M. Russel, and D.S. Miller. (1999). Active Lucifer Yellow
Ther. 289: 1104-1111.
22.
Masereeuw, R., F.G.M. Russel, and D.S. Miller. (1996). Multiple pathways of organic anion secretion in renal
23.
Masereeuw, R., S. A. Terlouw, R. A. M. H. van Aubel, F. G. M. Russel, and D. S. Miller. (2000). Endothelin B
receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule. Mol. Pharmacol., in
press.
24.
Muraoka, I., T. Hasegawa, M. Nadai, L. Wang, S. Haghgoo, O. Tagaya, and T. Nabeshima. (1995). Biliary
and renal excretions of cefpiramide in Eisai hyperbilirubinemic rats. Antimicrob. Agents Chemother. 39: 70
74.
25.
Murata, M., I. Tamai, Y. Sai, O. Nagata, H. Kato, Y. Sugiyama, and A. Tsuji. (1998). Hepatobiliary transport
kinetics of HSR-903, a new quinolone antibacterial agent. Drug Metab. Dispos. 26: 1113-1119.
26.
Niinuma, K., O. Takenaka, T. Horie, K. Kobayashi, Y. Kato, H. Suzuki, and Y. Sugiyama. (1997). Kinetic
analysis of the primary active transport of conjugated metabolites across the bile canalicular membrane:
Comparative study of S-(2,4-dinitrophenyl)-glutathione and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3pyridylmethyl) benzothiazole glucuronide. J. Pharmacol. Exp. Ther. 866-872.
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.
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 multidrugresistance 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.
82
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 beta
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).
glucuronide in the rat. Hepatology 23: 607-613.
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.
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.
83
Chapter 4
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,
Depts. o f 1Pharmacology and Toxicology, 2Biochemistry, and 3Cell Physiology,
Molecular Pharmacology, 1999, volume 56, pages 714-719
85
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 j M ) uptake into membrane vesicles from Sf9 cells infected with a
baculovirus encoding rabbit Mrp2 (Sf9-Mrp2) was similar to vesicles from mockinfected 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)
87
Chapter Four
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
C 4 (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 drugsensitive 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 CD DP [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], MRP2mediated 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
characterized by hyperbilirubinemia as well
lack functional
Mrp2,
as low biliary GSH
are
contents
compared to wildtype rats [13,24,30]. However, GSH uptake into EHBR liver
88
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
International (Buckinghamshire,
(15.5
UK).
Ci/mmol)
[14,
15,
was
purchased
from
Amersham
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 V BL 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
89
Chapter Four
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 TrisHepes, 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 TSbuffer 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 j g 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 j M
[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 j M
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).
90
TABLE 1
ATP-dependent uptake of [3H]VBL into membrane vesicles of Sf9-Mrp2 and
Sf9-mock cells
Compound
ATP-dependent [3H]VBL uptake
Sf9-Mrp2
Sf9-mock
pmol/mg/2 min
pmol/mg/2 min
5.0 ± 1.0
5.1 ± 1.2
±
0^0
.
Control
4.9 ± 0.5
23.1
GSH + MK571
12.0 ± 1 1a,b
5-methyl-GSH
15.2 ±
n.d.
0
.
GSH
4.6 ± 0.7
DTT
4.8 ± 1.2
5.3 ± 0.9
Glucuronate
4.9 ± 0.8
5.0 ± 1.2
Membrane vesicles were incubated in TS-buffer with 0.2
M
j
[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
W e investigated uptake of [3H]VBL into membrane vesicles prepared from Sf9 cells
infected with a baculovirus containing a rabbit mrp2 cDNA (Sf9-Mrp2). W e 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
91
Chapter Four
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.
Fig. 2. Osmolarity dependency of GSH-
stimulated ATP-dependent [3H]VBL
è l ic
E
25-
transport by Mrp2. GSH/ATP-dependent
[3H]VBL uptake into Sf9-Mrp2 membrane
(N
"ra 20Q E
z "o
LU E
CL 3
m
QI LU
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
CL
transport was plotted against the inverse
I— £
< CL
Ì 3
sucrose concentration in the reaction
CO
mixture. Linear fitting of the obtained data
0
was performed using GraphPad Prism
0
1
2
3
1/[SUCROSE] (1/M)
4
version 3.00 for Windows (GraphPad
Software, San Diego CA, USA). Data are
mean ± S.D. from one experiment
performed in triplicate.
92
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 timedependent 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 Sf9mock 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 ATPdependent [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 j M and Vmax of 89 + 6
pmol/mg/20 sec. W e 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.
93
Chapter Four
[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.).
W e investigated the effect of GSH and VBL on ATP-dependent uptake of the highaffinity substrate LTC4 into Sf9-Mrp2 membrane vesicles. As shown in Fig. 4, initial
rates of ATP-dependent [3H]LTC 4 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.
94
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.
VBL ^
+
no
GSH
“
+
0.1 mM
GSH
-
+
1 mM
GSH
-
+
5 mM
GSH
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, timedependent 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).
95
Chapter Four
Fig. 5. Time course and osmolarity
dependency of Mrp2-mediated [3H]GSH
transport. A and B, membrane vesicles
prepared from Sf9-Mrp2 (squares) and Sf9mock (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.
TIME (min)
1/[SUCROSE] (1/M)
96
Preloading of Sf9-Mrp2 membrane vesicles with 5 mM GSH reduced initial
[3H]GSH uptake rates by ~ 8 5 % either in the presence or absence of ATP (Table
2). Initial [3H]GSH uptake rates were also inhibited by LTC4, estradiol-17S-Dglucuronate (E2I 7 SG) and MK571 but only in the presence of ATP, whereas
S-methyl-GSH had no effect (Table 2).
TABLE 2
Effect of various compounds on [3H]GSH uptake into membrane
vesicles of Sf9-Mrp2 cells
[3H]GSH uptake
Compound
Control
+ATP
-ATP
100
100
GSH (5 mM, trans)
12
± 3a
S-methyl-GSH (5 mM)
98
± 5
LTC4 (5 ^M)
23
± 5a
97+17
E2 I 7 SG (0.1 mM)
50
+ 12a
89+12
+ 7a
MK571 (0.1 mM)
32
VBL (0.2 j j M)
9 8 +5
VBL (0.1 mM)
102+3
15
± 4a
100+2
9 9 + 7
98+10
107+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
97
Chapter Four
nmol/mg/min; n = 2) was not affected by either 0.2 j M , 10 j M 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 ATPdependent chloride channel CFTR, which belongs to the M RP branch of ABC98
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
99
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 Mrp2mediated 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 M RP2 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 D N A 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). c D N A 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 M RP. 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 D C K cells expressing c M O A T (MRP2) cD N A . J. Clin. Invest.
101: 1310-1319.
101
Chapter Four
8.
Fernandez-Checa, J. C., H. Takikawa, T. Horie, M. Ookhtens, and N. Kaplowitz (1992). Canalicular transport of
9.
Horio, M., M. M. Gottesman, and I. Pastan (1988). ATP-dependent transport of vinblastine in vesicles from
reduced glutathione in normal and mutant eisai hyperbilirubinemic rats. J. Biol. Chem. 267: 1667-1673.
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
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 M RP2. 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. W illiam s, 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 M RP2
(cM RP/cM OAT) gene in liver. FASEB J. 11: 509-516.
14. Koike, K., T. Kawabe, T. Tanaka, S. Toh, T. Uchiumi, M. W ada, S.-I. Akiyama, M. Ono, and M. Kuwano (1997).
A canalicular multispecific organic anion transporter (cM O AT) antisense c D N A 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 c M O A T (MRP2), M RP3, M RP4, and M RP5, homologous of the multidrug resistanceassociated 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). ATPdependent glutathione disulphide transport mediated by the M R P 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: 5130
5136.
21. Madon, J., U. Eckhardt, T. Gerloff, B. Stieger, and P. J. Meier (1997). Functional expression of the rat liver
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.
102
29. Renes, J., E. G. E. de Vries, E. F. Nienhuis, P. L. M. Jansen, and M. Müller (1999). ATP- and glutathionedependent transport of chemotherapeutic drugs by the multidrug resistance protein M R P 1 . 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
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 resistanceassociated protein. Proc. Natl. Acad. Sci. USA 92: 7690-7694.
103
Chapter 5
The Multidrug Resistance Protein Mrp2 M ediates ATPD ependent Transport of the Classical Renal O rganic Anion
p-Am inohippurate
Remon A.M.H. van Aubel1, Janny G.P. Peters1, Rosalinde Masereeuw1,
Depts. of Pharmacology and Toxicology and 2Cell Physiology,
Submitted for publication
105
106
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 brushborder (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 C 4 (K = 1.0 ± 0.4 jM ), estradiol175-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.
107
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 potentialdriven 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 brushborder 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 C 4
(LTC4), estradiol-175-D-glucuronide
(E2 1 7 SG),
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 j M ) or [3H]E2175G (50 nM) into membrane
vesicles was performed as described [28]. Briefly, membrane vesicles (40 j g proteinequivalent) 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).
109
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 Sf9mock 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
W e investigated uptake of [14C]PAH into membrane vesicles prepared from Sf9
cells infected with a baculovirus containing a full-length rabbit mrp2 cDNA (Sf9Mrp2). 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
(pmol/mg)
UPTAKE
Fig. 1. Time course and osmolarity
dependency of Mrp2-mediated [,4C]PAH
uptake. Membrane vesicles prepared from
[ 14C]PAH
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
(pmol/mg)
two experiments performed in triplicate.
C, ATP-dependent [14C]PAH uptake into
membrane vesicles of Sf9-mock (□) and Sf9Mrp2 (■) cells was determined for 5 min in
UPTAKE
ATP-DEPENDENT
[ 14C]PAH
membrane vesicles of Sf9-mock (□) and Sf9Mrp2 (■) cells. Data are the mean ± S.E. from
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
(pmol/mg/5 min)
in triplicate.
UPTAKE
ATP-DEPENDENT
[ 14C]PAH
mean ± S.D. from one experiment performed
1 /[s u c ro s e ] (1/M )
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 Sf9Mrp2 and Sf9-mock cells increased with increasing PAH concentrations (Fig 2A).
Values obtained using Sf9-Mrp2 membrane vesicles were corrected for basal ATPdependent [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 j M ) [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 j M , LTC4 inhibited Mrp2-mediated ATP-dependent [14C]PAH uptake to
basal levels observed in membrane vesicles of Sf9-mock cells.
W e 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. E2 1 7 SG, 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 j M 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.
113
Chapter Five
X
< Iz
Fig. 3. Effect of LTC4 on ATP-
150
2 1
dependent [14C]PAH uptake.
Membrane vesicles from Sf9-Mrp2
]
-Ï-
P £
£!
cells were incubated with 50 /j M
[14C]PAH for 5 min in the absence
100
or presence of 100, 250, 500, 1000,
LU O
Q E
LU
CL LU
m
Q <c
or 3000 nM LTC4 . Data are the
mean ± S.E. from two experiments
50
performed in triplicate. Line through
the data points is the fitted line
CL CL
according to a one-site competitive
model.
0
0 '
' 2
3
4
log [LTC4] (nM)
TABLE 1
Effect 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
% control
None (control)
E217£G (0.1 mM)
PAH (1 mM)
100
18 ± 8
nd
[3H]E217gG
% control
100
nd
65 ± 9
probenecid (0.1 mM)
90 ± 4a
39 ± 9
FL-MTX (10 yvM)
51 ± 9
38 ± 8
FURA-AM (10 yvM)
75 ± 2
3± 1
LY (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.
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 E2 1 7 SG (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 j M , which is consistent with a Km of approximately 1 j M 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 ATPdependent 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,
facilitated
diffusion
i.e. an anion exchange mechanism and potential-driven
[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 brushborder 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 FLMTX 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 ATPdependent 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 highaffinity 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
W e thank J.B. Koenderink (Dept. Biochemistry, University of Nijmegen) for expert
technical assistance.
116
References
1. Bergwerk, A.J., X. Shi, A.C. Ford, N. Kanai, E. Jacquemin, R.D. Burk, S. Bai, P.M. Novikoff, B. Stieger, P.J. Meier,
V.L. Schuster, and A .W . Wolkoff. (1996). Immunologic distribution of an organic anion transport protein in rat
liver and kidney. Am. J. Physiol. 271: G231-G238.
2. Cui, Y., J. König, U. Buchholz, H. Spring, I. Leier, and D. Keppler. (1999). Drug resistance and ATP-dependent
conjugate transport mediated by the apical multidrug resistance protein, M RP2, 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 membrane
4. Hosoyamada, M., T. Sekine, Y. Kanai, and H. Endou. (1999). M olecular cloning and functional expression of a
multispecific 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
6.
Kanai, N., R. Lu, Y. Bao, A .W . Wolkoff, and V.L. Schuster. (1996). Transient expression of oatp organic anion
transporter 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 M RP2
(cM RP/cM OAT) 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 conjugates
in 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 c M O A T is
functionally expressed on the apical membrane in Madin-Darby canine kidney (M D CK) 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. M iller. (1999). Active Lucifer Y ellow
Ther. 289: 1104-1111.
12. Masereeuw, R., F.G.M . Russel, and D.S. Miller. (1996). Multiple pathways of organic anion secretion in renal
13. Masereeuw, R., S. A. Terlouw, R. A. M. H. van Aubel, F. G. M. Russel, and D. S. M iller. (2000). Endothelin B
receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule. Mol. Pharmacol., in press.
14. Masuda, S., H. Saito, H. Nonoguchi, K. Tomita, and K.-I. Inui. (1997). m RN A 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. W ioland, 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.
117
Chapter Five
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 M RP2 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 G SSG and glutathione conjugates mediated by MRP1 and
cM O AT/M RP2. 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. Uw ai, 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 druginduced damage. J. Exp. Med. 188: 797-808.
118
Chapter 6
Summary,
General Conclusions and
Future Perspectives
119
12G
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 bilirubin
glucuronides.
The cloning of the gene encoding the protein responsible for cMOAT was
initiated from a different field of research.
Resistance of cancer cells to
121
Chapter Six
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 C 4 (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
122
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 ATPdependent 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 ATPdependently 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
123
Chapter Six
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 j M
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 ATPdependent [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,
E2 1 7 SG, 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).
124
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 j M , which is in close
proximity with its Km value. Mrp2-mediated ATP-dependent uptake of [14C]PAH
was strongly inhibited by E2 1 7 SG, whereas
[3H]E217SG
uptake only
modestly.
PAH
inhibited ATP-dependent
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 M DCK 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 tetracycline
regulatory expression system. Indeed, we obtained several M DCK 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. W e 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
125
Chapter Six
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 Mrp2mediated 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
126
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 ATPdependent 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.
127
128
Samenvatting en Conclusies
(Summary and Conclusions in Dutch)
129
130
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 (negatiefgeladen) 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.
131
Het erfelijke materiaal (gen) waarin de gegevens zijn opgeslagen voor het cMOATeiwit, 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-resistanceprotein (MRP) genaamd. Opmerkelijk was dat M RP 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 M RP erg veel op elkaar
lijken en aangezien er bovendien nieuwe MRP-genen (MRP3 t/m MRP6) zijn
gevonden, worden M RP 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
132
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 baculovirusgeïnfecteerde 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 ATPregenerend systeem dat het door Mrp2-verbruikte ATP opnieuw synthetiseert. W e
hebben laten zien dat konijnen-Mrp2 de twee lichaamseigen anionische stoffen
leukotrieen-C4 (LTC4) en estradiol-17-S-D-glucuronide (E2I 7 SG) ATP-afhankelijk
transporteert. Bij een ATP-concentratie van ongeveer 0.6 mM was het Mrp2gemedieerde transport van E2I 7 SG half-maximaal. Tenslotte hebben we laten zien
dat het ATP-afhankelijke E2I 7 SG- 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. W e 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-
133
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
E2 1 7 SG, 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 E2 1 7 SG 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). W e 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
134
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.
135
136
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.
137
138
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 Excelschema'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
139
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 coauteurschap 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. W e
zijn eerst getrouwd en hebben daarna het mooiste mannetje van de wereld
gekregen. Ik geniet er iedere dag van. Dankjewel.
140
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.
141

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