Báo cáo khoa học: cGMP and glutathione-conjugate transport in human erythrocytes The roles of the multidrug resistance-associated proteins, MRP1, MRP4 and MRP5 pot

cGMP and glutathione-conjugate transport in human erythrocytes
The roles of the multidrug resistance-associated proteins, MRP1, MRP4
and MRP5
Antonios Klokouzas†, Chung-Pu Wu, Hendrik W. van Veen, Margery A. Barrand and Stephen B. Hladky
Department of Pharmacology, University of Cambridge, UK
The nature of cGMP transport in human erythrocytes, its
relationship to glutathione conjugate transport, and pos-
sible mediation by multidrug resistance-associated proteins
(MRPs) have been investigated. MRP1, MRP4 and MRP5
are detected in immunoblotting studies with erythrocytes.
MRP1 and MRP5 are also detected in multidrug resistant
COR-L23/R and MOR/R cells but at greatly reduced levels
in the parent, drug sensitive COR-L23/P cells. MRP4 is
detected in MOR/R but not COR-L23/R cells. Uptake of
cGMP into inside-out membrane vesicles prepared by a
spontaneous, one-step vesiculation process is shown to be by
a low affinity system that accounts for more than 80% of the
transport at all concentrations above 3 l
M
. This transport
is reduced by MRP inhibitors and substrates including
MK-571, methotrexate, estradiol 17-b-
D
-glucuronide, and
S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by
glibenclamide and frusemide but not by the monoclonal Ig
QCRL-3 that inhibits high-affinity transport of DNP-SG by
MRP1. It is concluded that the cGMP exporter is distinct
from MRP1 and has properties similar to those reported
for MRP4. Furthermore the evidence suggests that the
protein responsible for cGMP transport is the same as

that mediating low-affinity DNP-SG transport in human
erythrocytes.
Keywords: multidrug resistance-associated protein; guano-
sine cyclic mono-phosphate transport; glutathione-conju-
gate transport; human erythrocytes; membrane vesicles.
Active transport of the cyclic nucleotide cGMP across
human erythrocyte membranes can be demonstrated using
intact cells [1] or inside-out membrane vesicle preparations
[2–4]. In the studies using inside-out membrane vesicles, the
active uptake of cGMP was found to be saturable with two
components, one of high-affinity (K
m
2–5 l
M
) [2,5] and
another of low-affinity (K
m
170 ± 50 l
M
)[5].Twocom-
ponents have also been described for the transport of
another organic anion, a glutathione conjugate S(2,4-
dinitrophenyl)glutathione (DNP-SG), in human erythro-
cytes [6,7].
Two members of the multidrug resistance-associated
protein (MRP) transporter family, MRP1 and MRP5 have
been detected previously in human erythrocyte membranes
[8,9], and transport by MRP1 has been conclusively shown
to account for the high-affinity component of DNP-SG
transport [10–12]. MRP4 and MRP5 have been shown to

transport the cyclic nucleotides, cAMP and cGMP [9,13,14]
and it has been suggested that MRP5 mediates the high-
affinity component of the cGMP transport [15]. However,
the same group has questioned this identification [16] and
recently it has been shown that when expressed in HEK293
cells, MRP4 and MRP5 mediate low-affinity transport of
cyclic nucleotides [17].
The aim of the present study was to investigate the nature
of cGMP transport in human erythrocytes, its relationship
to glutathione conjugate transport, particularly to the low-
affinity DNP-SG component, and its possible mediation by
MRP4 and/or MRP5. The present work provides evidence
from immunoblotting studies that both MRP5 [9] and
MRP4 are expressed in human erythrocytes. Using inside-
out membrane vesicles prepared by a spontaneous, one-step
vesiculation process, we identify a low affinity component
for the cGMP transport which accounts for more than 80%
of the transport at all concentrations above 3 l
M
.This
transport is reduced by a range of inhibitors and substrates
for MRPs including MK-571, methotrexate, E
2
17bG, and
DNP-SG and also by glibenclamide and frusemide. We
show that this cGMP exporter is distinct from MRP1 and
has characteristics similar to those reported for MRP4. The
evidence suggests that the protein responsible for cGMP
transport is the same as that mediating low-affinity
DNP-SG transport in human erythrocytes.

Correspondence to S. B. Hladky, Department of Pharmacology,
University of Cambridge, Cambridge, CB2 1PD, UK.
Fax: + 44 1223 334040, Tel.: + 44 1223 334019,
E-mail:
Abbreviations:ATP-c-S, adenosine 5¢-O-(3-thiotriphosphate);
DNP-SG, S(2,4-dinitrophenyl)glutathione; E217bG, estradiol 17-b-
D
-glucuronide; GSH, reduced glutathione; HRP, horseradish peroxi-
dase; IBMX, isobutylmethylxanthine; MK-571, (3-([[3-(2-[7-chloro-
2-quinolinyl]ethenyl)phenyl}-{(3-(dimethylamino-3-oxopropyl)-
thio}-methyl]thio) propanoic acid; MRP, multidrug resistance-asso-
ciated protein; PNGase F, peptide N-glycosidase F; Ro, 31–8220
bisindolylmaleimide; SITS, 4-acetamido-4¢-isothiocyano-2,2¢-disulf-
onic stilbene IX methanesulfonate.
Present address: Laboratory of Cell Biology, National Cancer
Institute, Building 37, Room 1B17, 37 Convent Drive, Bethesda,
MD 20892, USA.
Note: web page available at
(Received 20 May 2003, revised 14 July 2003, accepted 15 July 2003)
Eur. J. Biochem. 270, 3696–3708 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03753.x
Experimental procedures
Chemicals
[8-
3
H]cGMP (specific activity 13.9 CiÆmmol
)1
) was obtained
from Amersham Biosciences, [glycine-2-
3
H]GSH (specific

activity 40 CiÆmmol
)1
)and[
3
H]glibenclamide (specific acti-
vity 44.7 CiÆmmol
)1
) were obtained from New England
Nuclear, respectively.
M
5
I-1 mAb against MRP5 was a kind gift of R. J.
Scheper (Free University, Amsterdam, the Netherlands);
anti-MRP4 mAb was a kind gift of G. D. Kruh (Fox Chase
Cancer Centre, Philadelphia, PA, USA); QCRL-3 mAb was
purchased from Signet Laboratories, USA. M
5
I-1 and anti-
MRP4 mAbs have been previously described [18,19].
4-Acetamido-4¢-isothiocyano-2, 2¢-disulfonic stilbene
(SITS), adenosine 3¢,5¢-cyclic monophosphate (cAMP),
adenosine 5¢-O-(3-thiotriphosphate) (ATP-c-S), adenosine
triphosphate (ATP), 4-aminopyridine, aprotinin, 1-chloro-
2,4-dinitrobenzene, clotrimazole, creatine kinase, creatine
phosphokinase, daunorubicin, dideoxyforskolin, iso-
butylmethylxanthine (IBMX), doxorubicin, estradiol 17-
b-D-glucuronide, forskolin, glibenclamide, glutathione
(reduced form, GSH), glutathione S-transferase, guanosine
3¢,5¢-cyclic monophosphate (cGMP), imidazole, indometh-
acin, leupeptin, lithocholic acid 3-sulphate, methotrexate,

pepstatin A, probenecid, taurocholic acid, tetraethylammo-
nium chloride, Triton X-100, Tween 20, verapamil and
vincristine were all obtained from Sigma Chemicals. Calcein
was purchased from Molecular Probes. Staurosporine and
bisindolylmaleimide IX methanesulfonate (Ro 31–8220)
were obtained from Calbiochem. (3-([[3-(2-[7-chloro-2-qui-
nolinyl]ethenyl)phenyl}-{(3-(dimethylamino-3-oxopro-
pyl)-thio}-methyl]thio) propanoic acid, MK-571, was a
generous gift of M. Turner (Merck-Frosst Center for
Therapeutic Research, Quebec, Canada). Peptide N-glyco-
sidase F (PNGase F) was purchased from Promega.
Drugs were prepared in 10 m
M
Tris/HCl (pH 7.4) for
GSH, ATP, ATP-c-S, cGMP, taurocholic acid, imidazole,
vincristine and MK-571 or in 66% dimethyl sulfoxide/34%
water for glibenclamide, SITS, methotrexate, verapamil,
indomethacin, E
2
17bG and clotrimazole. The final concen-
tration of the dimethyl sulfoxide did not exceed 0.5% in
each experiment. GSH stock solutions (adjusted to pH 7.4)
were freshly prepared on the day of each experiment.
[
3
H]DNP-SG was synthesized enzymatically as previ-
ously described [12,20]. The purity of the
3
H-labelled DNP-
SG was determined by thin-layer chromatography on silica

gel plates [(0.25 · 40 · 80) mm, AlugramÒSIL G/UV254,
Macherey-Nagel, Germany] using n-propanol:water (7 : 3,
v/v) as solvent [21].
Cell lines
COR-L23/R and MOR/R are MRP1-overexpressing, multi-
drug-resistant, human large-cell lung tumour lines produced
by doxorubicin selection [22,23]. All cells were cultured on
plastic in growth medium containing RPMI-1640 medium
supplemented with 10% (v/v) foetal bovine serum, glutamine
(2 m
M
), penicillin (100 IUÆmL
)1
) and streptomycin
(100 lgÆmL
)1
) (complete RPMI-1640) in a 5% CO
2
humi-
dified incubator at 37 °C. The L23/R and MOR/R sublines
were maintained in the presence of 0.2 lgÆmL
)1
and
0.4 lgÆmL
)1
doxorubicin, respectively. The cells were kept
in drug-free medium for at least 48 h before use in
experiments. Cells were passaged when they became conflu-
ent. RNA protection assay of the doxorubicin-resistant
COR-L23/R and MOR/R cells [24] shows that these cells: do

not express P-glycoprotein; over-express MRP1 when com-
pared to the doxorubicin-sensitive controls; and express
MRP4 at a low level. MRP5 is expressed at very low level in
COR-L23 cells but at high level in MOR cells.
Preparation of inside-out human lung tumour cell
membrane vesicles
Membrane vesicles from human lung tumour cells were
prepared according to a method described previously [25]
in the presence of protease inhibitors (5 lgÆmL
)1
leupeptin,
2 lgÆmL
)1
aprotinin, 80 ngÆmL
)1
pepstatin A). Briefly, cells
were lysed in ice-cold hypotonic buffer (1 m
M
Tris/HCl,
pH 7.4) for 30 min at 4 °C. Following centrifugation
at 100 000 g for 30 min at 4 °C, the resulting pellet was
homogenized vigorously with a Teflon hand homogenizer in
buffer containing 10 m
M
Tris/HCl, 250 m
M
sucrose, and
protease inhibitors, layered over 38% (w/v) sucrose in
10m
M

Tris/HCl and centrifuged at 100 000 g for 30 min at
4 °C. The membranous material in the layer at the interface
with the sucrose was collected, washed and centrifuged at
100 000 g for 30 min at 4 °C. The resulting pellet was
re-suspended in transport buffer (10 m
M
Tris/HCl, 250 m
M
sucrose, pH 7.4), and stored in aliquots at )80 °C.
Preparation of inside-out human erythrocyte membrane
vesicles
Fresh venous blood was drawn from donors into tubes
containing EDTA or heparin and processed immediately.
There were five donors, each of whom gave informed
consent, two of northern European origin, one southern
European, one Chinese, and one Sri-Lankan. Membrane
vesicles were prepared by a spontaneous, one-step vesicu-
lation process as previously described [26–28] with minor
modifications. Briefly, red blood cells were washed three
times with 5 vols. of isotonic medium (80 m
M
KCl; 70 m
M
NaCl; 0.2 m
M
MgCl
2
;10m
M
Hepes; 0.1 m

M
EGTA,
pH 7.5). Higher concentrations of EGTA (0.5–3 m
M
)and
high pH (8.5) interfere with the vesiculation process [26].
The buffy coat and topmost cell layer were removed after
each wash. The packed cells were then lysed by addition to
90 vols. of ice-cold solution L (2 m
M
Hepes and 0.1 m
M
EGTA, pH 7.5) and subsequently centrifuged at 40 000 g
for 20 min at 4 °C. The supernatant was removed and the
pelleted ghosts were re-suspended in ice-cold solution L.
This step was repeated twice. After the last wash, the pellets
were re-suspended by addition of half the original packed
cell volume of cold solution L and incubated at 37 °Cfor
30 min resulting in spontaneous formation of spectrin-
actin-free vesicles [28]. After incubation, the suspension was
washed with solution L and the resulting pellet resuspended
in 10 m
M
Tris/HCl (pH 7.4). The protein concentrations of
the vesicle samples were determined using the BCA
(bicinchoninic acid) protein assay (Pierce). Membrane
vesicles were frozen and stored at )80 °C until use.
Ó FEBS 2003 cGMP transport in human erythrocytes (Eur. J. Biochem. 270) 3697
Measurement of membrane vesicle sidedness
The proportion of inside-out vesicles in the membrane

preparations was assessed by determining the accessibility of
the ectoenzyme acetylcholinesterase, and the endoenzyme
glyceraldehyde-3-phosphate dehydrogenase to their sub-
strates. Triton X-100 was used to disrupt the permeability
barrier and expose latent markers. The determination of
enzyme activities was performed colorimetrically [29,30].
The assays were modified by exchange of all phosphate
solutions with 10 m
M
Tris/HCl (pH 7.4) for the assays
involving membrane vesicles prepared from human eryth-
rocytes. The pH optimum of glyceraldehyde 3-phosphate
dehydrogenase activity is about 8.4 [31], but the activity in
the present study was determined at pH 7.4 to obtain
comparable conditions in the assays of sidedness and
transport. Generally 30–37% of the vesicles were inside-out.
Vesicle uptake studies
ATP-dependent uptake of radiolabeled cGMP or DNP-SG
into erythrocyte membrane vesicles was measured by a
rapid filtration technique [20]. Thawed membrane vesicles
were diluted in buffer and 50 lg protein added to a buffer
system (55 lL final volume) containing 1 m
M
ATP, 10 m
M
MgCl
2
,10m
M
creatine phosphate, 100 lgÆmL

)1
creatine
kinase, 10 m
M
Tris/HCl (pH 7.4) and 3.3 l
M
[
3
H]cGMP
or 3 l
M
[
3
H]DNP-SG or 254 l
M
[
3
H]DNP-SG. Aliquots
(20 lL) were taken from the mixture after 15 min in the case
of cGMP uptake, after 30 min with 3 l
M
[
3
H]DNP-SG
uptake,andafter45 minwith254 l
M
[
3
H]DNP-SG uptake,
diluted in 1 mL of ice cold stop solution (10 m

M
Tris/HCl,
pH 7.4) and subsequently filtered through nitrocellulose
filters (Whatman 0.2 lm pore size, presoaked overnight in
3% (w/v) bovine serum albumin. The filters were rinsed
with 3 mL of ice-cold stop solution and the tracer retained
on the filter was determined by liquid scintillation counting.
All transport data are presented as the difference between
the values measured in the presence of ATP and those
measured in the presence of the nonhydrolysable ATP
analogue, ATP-c-S. The ATP regenerating system (10 m
M
creatine phosphate, 100 lgÆml
)1
creatine kinase) was pre-
sent in both cases. Uptake of the substrate was expressed
relative to the protein concentration of the membrane
vesicles, and all data were corrected for the amount of
radiolabelled substrate bound to the filter in the absence of
vesicle protein. The substrate and inhibitor concentrations
are given in the respective figure legends. Tested compounds
were added from a stock solution in the appropriate solvent
[10 m
M
Tris/HCl (pH, 7.4), dimethyl sulphoxide or ethanol,
with the latter two solvents at a final concentration < 0.5%
v/v], identical concentrations of the vehicle being used in
control samples.
Curve fitting and statistics
Data are reported as mean ± s.e.m. Estimates of maximum

uptake rates and apparent dissociation constants were
obtained by least squares fits to the data using either the
solver in Microsoft
EXCEL
Ò or
KALEIDAGRAPH
Ò (Synergy
Software, Reading, PA, USA). Measurements of uptake vs.
substrate concentration were fitted assuming that transport
occurs as the sum of two processes each described by a Hill
equation [32]:
U ¼
U
max
c
n
K
n
D
þ c
n
; 1 n 2 ð1Þ
where c is the concentration, U
max
the maximum uptake,
K
d
the apparent dissociation constant and n is the Hill
coefficient, here expected to lie between 1 and 2. The data
were fitted to minimize the sum of squared proportional

deviations,
SSE ¼
X
i
U
i;observed
À U
i;fitted
ÀÁ
=U
i;fitted
ÀÁ
2
ð2Þ
fits to inhibition curves were to equations of the form:
U ¼
U
0
À U
noninh
ðÞÂIC
n
i
50
IC
n
i
50
þ I
n

i
þ U
noninh
; 1 ¼ n
i
¼ 2 ð3Þ
where I is the concentration of the inhibitor, IC
50
is the
inhibitor concentration producing 50% inhibition of the
inhibitable component, U
0
is the uptake in the absence of
the inhibitor, U
noninh
is the uptake which cannot be inhibited
and n
i
is the Hill coefficient for the inhibitor. For simple
competition, n
i
¼ 1. Unless stated otherwise, Kaleida-
GraphÒ was used to fit the data using the variances at
each concentration, r
i
, as the weights:
SSE ¼ v
2
¼
X

i
U
i;observed
À U
i;fitted
ÀÁ
=r
i
ÀÁ
2
ð4Þ
fits with different numbers of fitting parameters were
compared using an F-testontheratioofthevariance
associated with the reduction in degrees of freedom to the
variance of the fit with the smaller number of degrees of
freedom [33],
VR ¼
ðSSE
2
À SSE
1
Þ=ðd.f.
2
À d.f.
1
Þ
SSE
1
=d.f.
1

ð5Þ
where d.f. is the number of degrees of freedom (¼ number of
data points ) number of fitting parameters). The improve-
ment in fit is labelled ÔsignificantÕ if the probability from the
F-test is less than 0.05. Fits with the same number of degrees
of freedom were compared with each other using the
likelihood ratio:
LR ¼ exp À
DSSE
2r
2

ð6Þ
where DSSE is the difference in SSE for the two fits and the
noise variance was estimated as r
2
¼ MinSSE/d.f. with
MinSSE equal to the value of SSE for the best fit.
SDS/PAGE and Western blotting
Membrane vesicle or crude lysate proteins (5–40 lg) were
separated through 7.5% (w/v) polyacrylamide and subse-
quently transferred onto Hybond ECL nitrocellulose mem-
branes (Amersham Biosciences). Each membrane was
then incubated in blocking buffer [5% (w/v) milk powder
in 0.1% NaCl/P
i
/Tris/Tween (25 m
M
Tris/HCl, pH 7.4,
150 m

M
NaCl, 0.1% Tween 20)] overnight at 4 °Cprior
to the addition of the primary Ig (M
5
I-1, 1 : 40 dilution;
3698 A. Klokouzas et al.(Eur. J. Biochem. 270) Ó FEBS 2003
anti-MRP4, 1 : 300 dilution). The positions of the MRP
proteins on the membranes were visualized using the enhanced
chemiluminescence horseradish peroxidase (HRP) detection
system (Amersham Biosciences). The secondary antibodies
used were the HRP-conjugated rabbit anti-(rat IgG) Ig
(1 : 2000 dilution for M
5
I-1) or HRP-conjugated rabbit
anti-(mouse IgG) Ig (1 : 2000 dilution for anti-MRP4).
Membrane proteins from the human erythrocytes were
N-deglycosylated by treatment with PNGaseF as follows:
Briefly, membrane vesicles from human erythrocytes
(40 lg) were first denatured at 100 °C for 10 min in the
presence of 0.5% SDS and 1% b-mercaptoethanol, fol-
lowed by incubation at 37 °C for 1 h in the presence of
50 m
M
sodium phosphate (pH 7.5), 1% of the nonionic
detergent Nonidet P-40 (NP-40), and 2000 units of PNGase
F (New England Biolabs). PNGase F is an amidase which
cleaves between the innermost N-acetylglucosamine (Glc-
NAc) and asparagine residues of high mannose, hybrid and
complex oligosaccharides from N-linked glycoproteins [34].
PNGase F hydrolyzes nearly all types of N-glycan chains

from glycopeptides/proteins.
Results
ATP-dependent uptake of cGMP into human
erythrocyte membrane vesicles
The rate of ATP-dependent uptake of 3.3 l
M
[
3
H]cGMP at
37 °C into inside-out erythrocyte membrane vesicles was
approximately constant for more than 30 min at about
10pmolÆmg
)1
Æmin
)1
(Fig. 1A). Uptake of [
3
H]cGMP in the
absence of ATP but in the presence of the nonhydrolysable
ATP analogue, ATP-c-S, was less than 5% of the uptake
in the presence of ATP. The amount of [
3
H]cGMP taken
up by these vesicles was approximately twofold lower
(44 ± 3%, mean ± SEM, n ¼ 4) when measured with
NaCl/P
i
(140 m
M
NaCl; 3 m

M
KCl; 10 m
M
Na
2
HPO
4
;and
1.8 m
M
KH
2
PO
4
, pH 7.4) than with the usual low osmol-
ality transport buffer. Such a difference is to be expected as
the higher osmolality should decrease the volume of the
intravesicular space.
ATP-dependent uptake of[
3
H]cGMP was determined at
cGMP concentrations in the range 0.5–300 l
M
(Fig. 1B,C).
To test whether the uptake occurs via a single component,
the data were fitted assuming two components each
described by a Hill equation (see Eqn 1) as shown in
Fig. 1 and Table 1. The data imply that there is a large
(U
max

> 300 pmolÆmg
)1
Æmin
)1
), weakly cooperative (n %
1.1–1.4) low affinity component with dissociation constant,
K
d2
, in the range 50–85 l
M
and suggest that there may also
be a second, much smaller high affinity component with
K
d1
, in the range 0.5–2.5 l
M
. However, this latter compo-
nent, which may correspond to the uptake observed
previously [2,5], contributes less than 20% of the uptake
even for a low concentration, 3.3 l
M
,ofcGMP.
ATP-dependent uptake of DNP-SG into inside-out,
human erythrocyte vesicles
When inside-out erythrocyte membrane vesicles were incu-
bated at 37 °Cwith3l
M
[
3
H]DNP-SG, the uptake in the

presence of 1 m
M
ATP increased linearly in time for at least
Fig. 1. ATP-dependent uptake of cGMP into inside-out membrane
vesicles prepared from human erythrocytes. (Top) Uptake of 3.3 l
M
[
3
H]cGMP was measured in the presence of 1 m
M
ATP or the non-
hydrolysable analogue ATP-cS. (Middle) The variation of uptake rate
with concentration of cGMP. (Bottom) Haynes–Wolfe plot of the data
for low concentrations. In this type of plot a single, simple saturable
component of uptake (Hill coefficient ¼ 1) would yield a straight line.
The fitted constants for the curves in (Middle) and (Bottom) are given
in Table 1. The dotted curves are drawn for a single, simple saturable
component of transport (Hill coefficient ¼ 1); the dashed curves for a
single component described by a Hill equation with Hill coeffi-
cient ¼ 1.09, and the solid curve for two components, each described
by a Hill equation with Hill coefficients of 2 for the high affinity, low
capacity component and 1.3 for the low affinity, high capacity com-
ponent. Data for the four highest concentrations were determined in
three independent experiments from one preparation of vesicles. All
other data points represent at least three experiments and two vesicle
preparations.
Ó FEBS 2003 cGMP transport in human erythrocytes (Eur. J. Biochem. 270) 3699
60 min while uptake when ATP was replaced by ATP-c-S
was almost negligible [12]. ATP-dependent uptake of
[

3
H]DNP-SG in human erythrocyte vesicles was determined
over a broad concentration range (0.44–1000 l
M
) (Fig. 2).
As for cGMP, the data for DNP-SG were analyzed using a
two component Hill equation. The results of fits with several
different restrictive assumptions are shown in Table 2. The
quantitative fitting (variance ratio test) confirms, as is
obvious by eye, that the transport occurs via at least two
components. To explore the range of acceptable values of
the Hill coefficient for the low-affinity component, n
2
,least
squares fits were obtained for specified values of n
2
(Table 2). Acceptable fits (likelihood ratio ¼ 0.05 com-
pared to the best fit) were obtained for values of n
2
between
1 and 1.48. Over the range from 1 to 1.4, the low-affinity
dissociation constant decreases from 82 to 65 l
M
while that
for the high-affinity component varies from 0.5 to 2 l
M
.
In agreement with previous work [10,11] the high affinity
component of DNP-SG transport in these vesicles is most
likely mediated by MRP1 [12]. Strong evidence in support

of this comes from the observation that the uptake rate of
3 l
M
DNP-SG is reduced by at least 80% by QCRL-3 [12],
an MRP1-specific conformational-dependent monoclonal
Ig [35].
To investigate the low affinity component the DNP-
SG concentration was increased to 254 l
M
. The inhibi-
tion by QCRL-3 was then only 40 ± 5% (n ¼ 6). This
result and the complete inhibition observed at low DNP-
SG concentrations suggests that there is some low
affinity transport that is not inhibited by QCRL-3 and
is thus not mediated by MRP1. On this basis, the low
affinity process should account for no more than 20% of
the uptake observed at 3 l
M
. This requirement is
consistent with the uptake measurements provided
n
2
¼ 1.4. All the data are consistent with high affinity
transport via MRP1 (K
d
¼ 2 l
M
, U
max
¼ 20 pmolÆmg

)1
Æ
min
)1
) and low affinity, weakly cooperative transport via
a second transporter (K
d
¼ 65 l
M
, U
max
¼ 196 pmolÆ
mg
)1
Æmin
)1
and n
2
¼ 1.4).
Interrelations between cGMP and DNP-SG uptake
into human erythrocyte membrane vesicles
To explore the relations between cGMP and DNP-SG
transport, the ability of each to inhibit transport of the other
was investigated. ATP-dependent uptake of 3 l
M
DNP-SG
was not affected by the presence of cGMP at concentrations
up to 500 l
M
(Fig. 3A) suggesting that the high affinity

DNP-SG transporter, MRP1, does not transport cGMP.
Table 1. Fitting parameters for the uptake of [
3
H]cGMP into one-step,
inside out erythrocyte membrane vesicles shown in Fig. 1. The maximum
uptake rates, U
max1
and U
max2
, the dissociation constants, K
d1
and K
d2
,
and the Hill coefficients, n
1
and n
2
are defined as indicated in Eqn (1).
The data were obtained using two different vesicle preparations. As no
differences were observed between the two, the data were combined
without scaling. The residual value of the sum of squared proportional
deviations, SSE (see Eqn 2), is shown for each fit. For each column
except the first the variance ratio (see Eqn 5) has been calculated rel-
ative to the column immediately to the left. The fit obtained with the
constraints n
1
¼ 1 and n
2
¼ 1 is not shown as the fitted value of U

max1
was 0.000. These data imply (F-test on the variance ratio, P ¼ 0.0004)
that the low affinity component shows cooperativity, n
2
>1,andare
consistent with the presence of a high affinity component (F-test,
P ¼ 0.002), but do not specify its characteristics.
Fitted constant
Constraints
U
max1
¼ 0
n
2
¼ 1 U
max1
¼ 0 n
1
¼ 1 n
1
< ¼ 2
U
max1
/pmolÆmg
)1
Æ
min
)1
0 0 7.1 2.7
K

d1
/l
M
– – 2.35 0.674
n
1
––12
U
max2
/pmolÆmg
)1
Æ
min
)1
551 389 293 304
K
d2
/l
M
150 82 50 52
n
2
1.00 1.09 1.40 1.32
SSE 0.543 0.353 0.223 0.208
VR – 15.7 7.9 1.8
P 0.0004 0.002 0.187
Fig. 2. Rate of ATP-dependent uptake of [
3
H]DNP-SG into inside-out
erythrocyte membrane vesicles. The dotted curve is drawn for a single

simple saturable component of uptake, the solid curve for two simple
saturable components. The dashed and dash-dot curves are drawn for
two components each obeying a Hill equation with the constraints that
n
2
¼ 1.4 or n
2
¼ 2, respectively. The data are plotted directly (Top)
and as a Haynes–Wolfe plot (Bottom). The fitted constants are des-
cribed in Table 2. These data are not consistent with a single-compo-
nent of uptake, but cannot unambiguously determine the properties of
two components when provision is made for the possibility that more
than one substrate molecule may interact with the transporter at a
time.
3700 A. Klokouzas et al.(Eur. J. Biochem. 270) Ó FEBS 2003
This finding was further supported by the observation that
the MRP1-specific Ig, QCRL-3, produced negligible inhi-
bition of the ATP-dependent uptake of cGMP (uptake
rate for 3.3 l
M
cGMP with 10 lgÆmL
)1
QCRL-3 was
98 ± 5% of control, n ¼ 3).
On the other hand, ATP-dependent uptake of DNP-SG at
high concentrations was inhibited by cGMP (Fig. 3B). The
fitted curve for uptake at 254 l
M
DNP-SG has two
components: a noninhibitable uptake (30 min) of 2943 ±

115pmolÆmg
)1
and a component of 4110 ± 144 pmolÆmg
)1
inhibitable by cGMP with an IC
50
of 133 ± 18 l
M
.These
components are plausibly attributed to the high and low-
affinity components of DNP-SG transport, respectively.
In order to investigate whether the cGMP transport is also
affected by increasing concentrations of DNP-SG, uptake of
3.3 l
M
[
3
H]cGMP in inside-out membrane vesicles was
measured in the presence of DNP-SG in the range of 0.5–
800 l
M
(Fig. 3C). DNP-SG was able to inhibit all of the
cGMP transport detectable at this concentration suggesting
that it occurs via a single, DNP-SG inhibitable component.
The solid curve is a plot of a Hill equation (see Eqn 3) with
IC
50
82 ± 2 l
M
and a Hill coefficient of 1.25 ± 0.02 l

M
.
Effect of MRP inhibitors, substrates, and modulators on
cGMP uptake into human erythrocyte membrane vesicles:
A number of compounds that are known to interact with
one or more MRPs were tested for their ability to inhibit
cGMP transport (see Fig. 4 and section below entitled
ÔcGMP transport is inhibited by anion transport inhibitors,
PKC inhibitors and IBMXÕ). MK-571, a leukotriene D
4
(LTD
4
) receptor antagonist, which has been shown to
inhibit transport by MRP1 [36,37], MRP2 and MRP3 [38]
and MRP4- [39] but not MRP5-mediated cGMP transport
[9] completely inhibited the [
3
H]cGMP uptake in vesicles
with an IC
50
value of 0.38 ± 0.01 l
M
(Fig. 4A) suggesting
that this transport is not mediated by MRP5. cAMP
(Fig. 4B) which is transported by MRP4 and MRP5 [9,13],
inhibited with IC
50
¼ 296 ± 26 l
M
. The ratio of this

constant to the apparent dissociation constant for cGMP,
50–80 l
M
(Table 1), is similar to the ratio, 6, for MRP4 [13],
but is much smaller than the ratio, 380, for MRP5 [9].
Glibenclamide (Fig. 4C), an agent known to bind to various
ABC proteins [40,41] including the sulphonylurea receptor
[42,43], was effective in inhibiting the cGMP transport in
human erythrocyte vesicles at micromolar concentrations.
Substantial inhibition was produced by methotrexate and
E
2
17bG, established MRP4 substrates, by indomethacin
which is known to inhibit transport by MRP1 and MRP2
[44], and by clotrimazole (Fig. 4D) an imidazole-derived
antifungal agent which inhibits MRP1 mediated transport
[12]. Imidazole, the backbone molecule of clotrimazole had
no effect. Taurocholic acid, an established substrate for
MRP1, MRP2, and MRP3, inhibited but only at concen-
trations sufficiently high (> 200 l
M
, Table 3) that it may be
acting in a Ôdetergent likeÕ manner.
Reduced glutathione (GSH, pH 7.4), in the range of
0.5–4 m
M
, neither enhanced nor inhibited cGMP uptake
(Table 3). This contrasts with the effect of 1–5 m
M
GSH to

stimulate uptake of DNP-SG in human erythrocyte vesicles
[12] but is consistent with the lack of effect of GSH on
MRP4 and MRP5 mediated transport in transfected
HEK293 cells [17].
The cationic vinca alkaloid, vincristine, and the organic
anion, calcein, which are established MRP1 substrates, were
also tested for their ability to inhibit cGMP uptake. Calcein
inhibited cGMP uptake (about 25% inhibition at 100 l
M
and about 60% at 300 l
M
) but vincristine itself had no
effect even at 200 l
M
(Table 3). It is known that several
cationic MRP1 substrates, including vincristine, require
GSH for their transport and that vincristine inhibits the
high affinity DNP-SG transport in the presence but not in
the absence of GSH [12,45,46]. Thus the effect of vincristine
on the cGMP uptake was also tested in the presence of
Table 2. Fitting parameters for uptake of [
3
H]DNP-SG into one-step, inside out erythrocyte membrane vesicles. The maximum uptake rates, U
max1
and U
max2
, the dissociation constants, K
d1
and K
d2

,andtheHillcoefficients,n
1
and n
2
are as defined in Eqn (1). The data were collected in three
series using different vesicle preparations. To allow simultaneous fitting of all three sets of data, all data in the first set are scaled by multiplication by
AF and all data in the second by CF. For fits of the two component Hill equation, there are 22 remaining degrees of freedom. The one and two
component fits are compared with each other using an F-test on the variance ratio (Eqn 5). The two component fit is significantly better. The
various constrained two component fits are compared with the fit for n
1
> ¼ 1, n
2
> ¼ 1 using the likelihood ratio (Eqn 6). These data are
consistent with any value of n
2
between1and1.48(LR¼ 0.05).
Fitting constant
Constraints
U
max1
¼ 0;
n
2
> ¼ 1
n
1
> ¼ 1;
n
2
> ¼ 1

n
1
> ¼ 1;
n
2
> ¼ 1.2
n
1
> ¼ 1;
n
2
> ¼ 1.4
n
1
> ¼ 1;
n
2
> ¼ 2
U
max1
/pmolÆmg
)1
Æmin
)1
0 6.4 13.5 19.5 32.0
K
d1
/l
M
– 0.52 1.32 1.99 3.62

n
1
– 1.00 1.00 1.00 1.00
U
max2
/pmolÆmg
)1
Æmin
)1
197 235 212 196 171
K
d2
/l
M
36 82 71.554 65 56
n
2
1.00 1.00 1.20 1.40 2.00
AF 0.643 0.709 0.703 0.702 0.705
CF 0.110 0.108 0.108 0.108 0.109
SSE 2.155 0.476 0.500 0.529 0.611
VR 25.9
P 2 · 10
)7
LR 1 0.34 0.088 0.002
Ó FEBS 2003 cGMP transport in human erythrocytes (Eur. J. Biochem. 270) 3701
1m
M
GSH but no inhibition was observed. Given that
MRP1-mediated transport of calcein in whole cells can be

modulated by the level of GSH present [47], the effect of
calcein on cGMP uptake into erythrocyte membrane
vesicles was also tested in the presence of 1 m
M
GSH but
no additional inhibition was observed (Table 3).
These results are all consistent with cGMP transport via
MRP4 while the strong inhibition produced by MK-571
and the relative potency of cAMP appear to be incompati-
ble with transport via MRP5.
CGMP transport is inhibited by anion transport
inhibitors, PKC inhibitors and IBMX
Because substrates for MRPs are often organic anions,
inhibitors that block ion transport were tested (Table 4).
The anion transport inhibitors frusemide, niflumic acid,
phloridzin, SITS and probenecid all reduced the rate of
cGMP uptake (Table 4). By contrast the potassium channel
blockers, 4-aminopyridine, tetraethylammonium chloride
and CsCl, had no observable effect though BaCl
2
did
(Table 4). Verapamil, a calcium channel blocker, an inhi-
bitor of P-glycoprotein, and a general though weak inhibitor
of MRPs in vesicular drug uptake studies, reduced cGMP
transport in the presence or absence of 1 m
M
GSH. Two
protein kinase C inhibitors, staurosporine and Ro 31–8220
[48], were also tested for their ability to block cGMP
transport in human erythrocytes. Staurosporine has recently

been shown to bind directly to several ABC transporters [49]
in addition to preventing phosphorylation of these trans-
porters in intact cells [50]. Staurosporine at 10 l
M
com-
pletely inhibited the cGMP uptake while Ro 31–8220 at
10 l
M
showed only weak inhibition. Forskolin, an activator
of adenylyl cyclase, inhibited while its inactive analogue,
1,9-dideoxyforskolin, had no effect at the same concentra-
tion. IBMX which is structurally related to cGMP and
currently used as a nonspecific phosphodiesterase inhibitor,
inhibited transport. All of these effects are compatible with
cGMP transport by a member of the MRP family.
Immunodetection of MRP4 and MRP5 proteins
in human erythrocytes and COR-L23/R cells
To identify candidate proteins that could possibly mediate
the cGMP transport, immunoblot analysis was performed
on membrane vesicles from human erythrocytes using
monoclonal antibodies against MRP5 [18] and MRP4 [19].
The anti-MRP5 Ig, M
5
I-1, specifically detected an intact
band at 190 kDa which shifted to 160 kDa after treatment
with peptide N-glycosidase F (PNGaseF) (Fig. 5A) suggest-
ingthatMRP5isN-glycosylated. A protein with the same
apparent molecular mass was also detected in doxorubicin-
resistant MOR/R and COR-L23/R lung tumour cells but at
greatly reduced level in the doxorubicin-sensitive COR-L23/

P lung tumour cells (Fig. 5B). The anti-MRP4 Ig detected an
intact band at 170–180 kDa in human erythrocytes and
MOR/R cells but not in COR-L23/R cells (Fig. 5B).
Discussion
It is now well recognized that inside-out membrane vesicles
prepared from human erythrocytes can take up both
Fig. 3. Effect of cGMP on the DNP-SG transport in human erythro-
cytes and vice versa. (Top) ATP-dependent uptake of 3 l
M
[
3
H]DNP-
SG (30 min at 37 °C) was not affected by cGMP at concentrations up
to 500 l
M
. (Middle) ATP-dependent uptake of 254 l
M
[
3
H]DNP-SG
(30 min at 37 °C) was partially inhibited by cGMP. The fitted curve
corresponds to two components: a noninhibitable component of
2943 ± 115 pmolÆmg
)1
and a component of 4110 ± 144 pmolÆmg
)1
inhibitedbycGMPwithanIC
50
of 133 ± 18 l
M

(mean ± SEM).
(Bottom) ATP-dependent uptake of 3.3 l
M
[
3
H]cGMP (15 min at
37 °C) was inhibited by DNP-SG as a single component described by
a Hill equation with U
0
¼ 123 ± 1 pmolÆmg
)1
,IC
50
¼ 82 ± 2
pmolÆmg
)1
, and a Hill coefficient of 1.25 ± 0.02 (v
2
¼ 208). The fit of
the Hill equation was significantly better (variance ratio test, 17 data
points, three parameters in the Hill equation, P ¼ 0.003) than the
fit to a simple competition curve (U
0
¼ 135 ± 1 pmolÆmg
)1
,
IC
50
¼ 47 ± 1 pmolÆmg
)1

, v
2
¼ 395).
3702 A. Klokouzas et al.(Eur. J. Biochem. 270) Ó FEBS 2003
glutathione-conjugates, such as DNP-SG, and cyclic nucleo-
tides, e.g. cGMP, by rapid ATP-dependent transport
processes. In the present study, uptake of cGMP is shown
to consist primarily of a low-affinity component with a
maximum uptake (U
max
) of 300–400 pmolÆmg
)1
Æmin
)1
and
a dissociation constant (K
d
) in the range of 50–82 l
M
and
possibly also a second high affinity component of uptake
contributing less than 20% of the total trasnport even at low
concentrations. However, MK-571, glibenclamide, DNP-
SG, clotrimazole and cAMP all inhibit this uptake as if
there were only a single component of transport (Figs 3C
and 4). If present, the high-affinity component may
correspond to the high-affinity transport previously repor-
ted. In those studies [2,5] ATP-dependent cGMP uptake
into inside-out membrane vesicles from human erythrocytes
was found to have two components, a high affinity uptake

with U
max1
¼ 0.2–0.4 pmolÆmg
)1
Æmin
)1
and K
d1
¼ 2.4–
4.7 l
M
and a low affinity uptake with U
max2
¼ 1.6
pmolÆmg
)1
Æmin
)1
and K
d2
¼ 170 l
M
. The maximum up-
take rates in these studies were very low, being just over
threefold higher than the background [2]. The maximum
uptake rate in the current study is two orders of magnitude
higher. The reasons for this remarkable difference are
unclear though there may be several factors involved. These
include different osmolalities of the solutions used to
measure uptake, differences in the methods of vesicle

preparation, and possibly even differences in the profile of
transporters present on the red cell membranes from
different donors.
In the present study, the vesicles were resealed and
assayed in low osmolality solution. This contrasts with the
previous study where the vesicles were resealed at low
Fig. 4. Inhibition of uptake of 3.3 l
M
cGMP by (A) MK-571, (B) cAMP, (C) glibenclamide and (D) clotrimazole. (A) Inhibition by MK-571. The
curve is the best fit assuming simple competition, U
0
¼ 130 ± 2 pmolÆmg
)1
,IC
50
¼ 0.38 ± 0.01 l
M
, v
2
¼ 95. The Hill equation provides a closer
fit with, U
0
¼ 120 ± 2 pmolÆmg
)1
,IC
50
¼ 0.48 ± 0.02 l
M
, n
i

¼ 1.10 ± 0.02 and v
2
¼ 70, but the improvement is not significant (F ¼ 2.7,
P ¼ 0.13). The best fit allowing for noninhibitable uptake assigns a negative value ()1.4 pmolÆmg
)1
) to the noninhibitable component.
(B) Inhibition by cAMP. The curve shows the best fit for simple competition with U
0
¼ 108 ± 5 pmolÆmg
)1
,IC
50
¼ 296 ± 26 l
M
, v
2
¼ 11.Fits
of the Hill equation and of simple competition plus a noninhibitable component of uptake are almost superimposed on that shown (and
improvements in fit were not-significant with F and P-values for the variance ratio relative to simple-competition of 0.03 and 0.86, and 0.24 and
0.64, respectively). (C) Inibition by glibenclamide. The curve is the best fit for simple competition U
0
¼ 80 ± 1 pmolÆmg
)1
,IC
50
¼ 2.8 ± 0.1 l
M
,
v
2

¼ 250. Fits of the Hill equation and of simple competition plus a noninhibitable component of uptake did not produce significant improvements
in the fit (F and P-values 3.26 & 0.12 and 5.0 & 0.07, respectively). The best fit for the noninhibitable component was 3.4 ± 0.3 pmolÆmg
)1
.
(D) Inhibition by clotrimazole. The curve is the best fit for simple competition U
0
¼ 97 ± 1 pmolÆmg
)1
,IC
50
¼ 24 ± 1 l
M
, v
2
¼ 86. Fits of the
Hill equation and of simple competition plus a noninhibitable component did not produce significant improvements in the fit (F and P-values of 4.4
& 0.1 and 4.7 & 0.1, respectively. The best fit value of the Hill coefficient was less than 1 (0.72).
Ó FEBS 2003 cGMP transport in human erythrocytes (Eur. J. Biochem. 270) 3703
osmolality but assayed in NaCl/P
i
with an osmolality
10–100-fold higher. To check these solutions as possible
influencing factors, cGMP uptake in the current study was
also measured using solutions as employed in the previous
studies. Under these conditions, uptake rates were lower,
but only by a factor of two, i.e. too small an effect to explain
alone the discrepancies in results between the two studies.
Another possible explanation for the discrepancy is the
presence of traces of calcium in the previous studies. It was
shown there that calcium can inhibit cGMP transport with

an IC
50
of about 40 l
M
and the inclusion of 100 l
M
EGTA
was found to increase uptake rates by 100% [4] Even so no
chelators were included in most of those studies either
during vesicle preparation or during uptake measurements.
The vesicle preparations used in the present work were
produced by a one-step spontaneous vesiculation method in
the presence of 100 l
M
EGTA [26–28].
Further differences relate to the percentage of inside-out
vesicles generated. The previous study used the procedure of
Steck and Kant [30] which yields a much higher percentage
(typically > 60%) of inside-out vesicles than are routinely
produced by spontaneous vesiculation (usually 30–37%).
However, these differences would be expected to produce
lower uptake rates in the vesicles generated by spontaneous
vesiculation, not higher rates as observed here. Whether
there are inhibitory elements on the vesicle membranes that
are stripped off more effectively by spontaneous vesicula-
tion or stimulatory elements removed in the lengthy Steck
and Kant procedure remains to be determined. Certainly it
has been proposed that the spontaneous vesiculation
process may remove restrictive links with cytoskeletal
elements [28] allowing more lateral mobility and thus the

possibility of alterations in associations between membrane
proteins. The additional possibility that different donors
possess different profiles of transporters on their red cell
membranes is currently being explored.
In contrast to uptake of cGMP, ATP-dependent uptake
of DNP-SG into inside-out erythrocyte membrane vesicles
in the current study clearly possesses more than one
component. This has been noted also by Akerboom et al.
[7] and Pulaski et al. [10]. The observations are consistent
with the presence of two components, one, a low-capacity
Table 3. Effect of MRP substrates, inhibitors and modulators on the
ATP-dependent uptake of [
3
H]cGMP by inside-out human erythrocyte
membrane vesicles. Data represent mean ± SEM of n experiments. The
control uptake of [
3
H]cGMP (addition of dimethylsulfoxide only) for
indomethacin, methotrexate and E
2
17bG was 77.3 ± 5.2 pmolÆmg
)1
.
The control uptake for the remaining drugs was 136.5 ± 2 pmolÆ
mg
)1
.
Compound
Concentration
(l

M
)
[
3
H]cGMP uptake
(% control) n
GSH 500 101.2 ± 0.8 3
1000 108.3 ± 7.1 6
2000 100.1 ± 1.9 3
4000 98.0 ± 1.2 3
Vincristine 100 106.3 ± 7.6 3
200 100.6 ± 3.2 3
+1m
M
GSH 100 116.4 ± 1.1 3
Calcein 100 73.9 ± 1.8 3
300 38.5 ± 3.4 3
+1m
M
GSH 100 74.4 ± 1.8 3
Indomethacin 20 95.9 ± 3.7 3
50 7.9 ± 5.6 3
Methotrexate 275 25.2 ± 2.8 3
375 3.5 ± 1.1 3
17bE
2
G 65 12.6 ± 3.7 4
Taurocholic acid 200 80.1 ± 2.1 3
350 43.1 ± 1.1 3
Table 4. Effect of ion channel inhibitors on the ATP-dependent uptake

of [
3
H]cGMP by inside-out human erythrocyte membrane vesicles. Data
represent mean ± SEM of n experiments. The control level of
[
3
H]cGMP uptake with dimethylsulfoxide (n ¼ 16) for furosemide,
SITS, probenecid, staurosporine and Ro 31–8220 was in the range of
77–82 pmolÆmg
)1
protein. The control level of [
3
H]cGMP uptake with
ethanol (n ¼ 18) for phloridzin, niflumic acid, and verapamil was in
the range of 90–105 pmolÆmg
)1
protein. The control level of
[
3
H]cGMP uptake for the remaining drugs was in the range of 129–
135 pmol mg
)1
protein.
Compound
Concentration
(l
M
)
[
3

H]cGMP
uptake
(%control) n
Anion channel inhibitors
Frusemide 0.5 95.3 ± 2.3 6
1 90.7 ± 3.1 6
5 37.1 ± 2.1 8
10 19.4 ± 2.6 6
20 6.6 ± 0.9 4
50 6.9 ± 1.3 9
Niflumic acid 1 105.2 ± 2.1 4
5 45.6 ± 3.7 6
10 21.4 ± 1.5 6
20 5.4 ± 0.5 3
50 5.6 ± 1.1 9
Phloridzin 1 109.8 ± 3.3 5
5 99.9 ± 4.7 5
10 75.2 ± 4.2 7
50 28.3 ± 2.3 10
SITS 200 3.1 ± 0.9 3
Probenecid 200 11.3 ± 0.7 3
400 8.1 ± 1.1 3
Cation channel inhibitors
BaCl
2
400 64.7 ± 3.2 3
1000 47.9 ± 1.6 3
CsCl 400 100.1 ± 2.7 3
1000 102.4 ± 3.1 3
4-Aminopyridine 500 87.7 ± 4.6 3

Tetraethylammonium Cl 10000 87.3 ± 3.4 3
Verapamil 50 66.8 ± 1.7 3
Verapamil + 1 m
M
GSH 50 71.3 ± 3.5 3
PKC inhibitors
Staurosporine 10 2.9 ± 0.7 3
Ro31–8220 10 74.2 ± 2.3 3
Various
Colchicine 50 98.4 ± 3.0 8
Imidazole 100 94.1 ± 0.8 3
Dideoxyforskolin 100 95.3 ± 2.2 3
Forskolin 100 66.7 ± 1.6 3
IBMX 100 16.5 ± 1.3 4
3704 A. Klokouzas et al.(Eur. J. Biochem. 270) Ó FEBS 2003
high-affinity component (K
d
1–10 l
M
) that predominates
at concentrations below 5 l
M
and another low-affinity
component (> 100 l
M
)thatisdominantathighconcen-
trations.
The high-affinity component of DNP-SG transport,
identified previously as being mediated by MRP1 [10–12],
appears not to be involved in transport of cGMP (see

below). However, the low affinity component of DNP-SG
transport is inhibited by cGMP and may well be the
transporter that mediates low-affininty cGMP uptake.
DNP-SG can indeed inhibit cGMP transport. Furthermore,
the apparent dissociation constant for DNP-SG uptake,
k
d2
,andtheIC
50
for DNP-SG-mediated inhibition of
cGMP uptake are approximately equal, i.e. 65 l
M
com-
paredwith82 l
M
. Though the two values for cGMP uptake
and for cGMP inhibition of DNP-SG uptake differ, i.e. 50–
82 l
M
compared with 133 l
M
, it remains possible that a
single transporter accounts for both uptakes as the inter-
action between cGMP and DNP-SG is likely to be more
complex than simple competition. Further evidence for
greater complexity is provided by the curve fits with Hill
coefficients greater than 1.
As a wide variety of MRP transport inhibitors block
cGMP and DNP-SG transport (Table 3 and Fig. 4),
members of the MRP family are very plausible candidates

for the transporters mediating ATP-dependent uptake of
cGMP and DNP-SG into inside-out erythrocyte vesicles.
MRP1 and MRP5 have previously been detected on the
membranes of human erythrocytes [8,9]. MRP1 is known to
mediate the high-affinity transport of DNP-SG [10–12] and
MRP4 and MRP5 are known to transport cGMP [9,13,17].
In the present study in addition to confirming the presence
of MRP1 and MRP5 in erythrocyte membranes, we have
detected the presence of MRP4 (see Fig. 5). Although no
cross-reactivity tests have been reported for anti-MRP4, the
present finding that the anti-MRP4 Ig does not detect an
immunoreactive band in COR-L23/R cells which express
MRP1 and MRP5 suggests that this Ig does not cross-react
with MRP1 or MRP5.
Several lines of evidence show that MRP1 is most
unlikely to be the transporter responsible for the transport
of cGMP. It has already been shown that cGMP does not
inhibit transport of LTC
4
, an established high-affinity
MRP1 substrate [4]; cGMP does not inhibit MRP1-
mediated transport of DNP-SG; and, verapamil inhibits
cGMP uptake without any requirement for GSH (this
study) while GSH is needed for its inhibition of MRP1-
mediated transport [12,51]. Most convincingly, uptake of
3.3 l
M
cGMP is unaffected by the presence of the confor-
mation-dependent monoclonal Ig against MRP1, QCRL-3,
at 10 lgÆmL

)1
whichhasbeenshowntoblockMRP1
mediated high-affinity uptake of DNP-SG [12].
Both MRP4 and MRP5 have been shown to transport
cGMP, though the apparent dissociation constants have
proven controversial. The original reports indicated relat-
ively high-affinity trasnport with K
m
values in the range of
2–10 l
M
[9,13], but the transport is clearly much lower
affinity in HEK293 cells transfected with MRP4 or MRP5
[17]. To examine further the possible role of the MRPs in
erythrocytes, several established MRP substrates, inhibitors,
and modulators were tested for their ability to block the
[
3
H]cGMP uptake into inside-out vesicles. MK-571, a
leukotriene receptor antagonist, inhibited the cGMP trans-
portwithanIC
50
value of 0.38 ± 0.01 l
M
. This appeared
to be the most potent of the inhibitors tested in the present
study. MK-571 has been shown to inhibit MRP1, MRP2,
MRP3 and MRP4 mediated transport [9,36,38,39] but has
been reported to have no affect at concentrations of up to
50 l

M
on cGMP transport attributed to MRP5 [9].
Methotrexate and E
2
17bG were found to inhibit the
cGMP uptake in human erythrocyte vesicles with E
2
17bG
at 65 l
M
inhibiting about 90% and methotrexate inhi-
biting about 75% at 275 l
M
and completely at 375 l
M
.
These compounds are established MRP4 substrates with
K
m
values around 220 l
M
for methotrexate [19,39], and
30 l
M
for E
2
17bG [39,3,4]. At present, methotrexate and
E
2
17bG appear to be substrates of MRP4 [13,39] and not

of MRP5.
cAMP, which has been shown to be transported by both
MRP4 and MRP5 [9,13], inhibited all of the cGMP uptake
with an estimated IC
50
value of 315 ± 70 l
M
.Theratioof
this value to the apparent K
d
value for cGMP, about 5, is
Fig. 5. Immunodetection of MRP4 and MRP5 in human erythrocytes
and COR-L23 cells. Inside-out membrane vesicles prepared from
human erythrocytes, COR-L23/P and COR-L23/R cells, and crude
lysates from MOR/ADR cells were size fractionated on 7.5% SDS/
PAGE, blotted and immunostained with (A) M
5
I-1 mAb, and
(B) anti-MRP4 mAb, detecting MRP5 and MRP4, respectively.
Membranes (40 lg) from human erythrocytes were also treated with
PNGase F to remove the N-glycans and then immunostained with
M
5
I-1 (A). The amount of protein loaded per lane is indicated at the
bottom of each blot. Arrows mark the immunodetected band for
MRP4 and MRP5. Doxorubicin-resistant lung adenocarcinoma cell
line MOR/R was used as a positive control; doxorubicin-sensitive
human large-cell lung tumour cell line COR-L23/P was used as a
negative control.
Ó FEBS 2003 cGMP transport in human erythrocytes (Eur. J. Biochem. 270) 3705

consistent with the value, 5, reported for MRP4 [13] but not
the value, 380, reported for MRP5 [9].
Taurocholic acid, an established substrate for MRP1,
MRP2, and MRP3, had only a weak inhibitory effect on the
cGMP uptake (about 20% at 200 l
M
and about 60% at
350 l
M
). Interpretation of such inhibition is difficult given
that taurocholic acid would probably be acting as a
detergent at high concentrations.
Glibenclamide was found to be a very potent inhibitor of
cGMP transport in the human erythrocyte membrane
vesicles with an estimated IC
50
value of 1.9 ± 0.1 l
M
.This
was the second most potent of the inhibitors tested against
the cGMP transporter. Glibenclamide has been shown to
interact with several ABC proteins including the sulfonyl-
urea receptor [42,52], CFTR [40], and more recently,
P-glycoprotein and MRP1 [41,53], blocking their function.
Given that glibenclamide inhibits many ABC transporters,
its action serves merely to point to an ABC transporter as
being responsible for cGMP transport but not to identify
which ABC transporter it is.
Several other compounds including forskolin, an activa-
tor of adenylate cyclase, and IBMX, a nonspecific phos-

phodiesterase inhibitor with structural similarity to cGMP,
also inhibited the cGMP uptake. Forskolin at a tested
concentration of 100 l
M
inhibited the cGMP transport by
about 35% while its inactive analogue, 1,9-dideoxyforsko-
lin, had no effect at the same concentration. Forskolin has
previously been shown to inhibit the cGMP transport in
other types of erythrocyte vesicle preparations [2] but the
exact mechanism of its action remains unknown. It is
possible it could interact with a drug-binding site on the
cGMP transporter which bears structural homology to
adenylyl cyclase, as it has been previously proposed for the
effect of forskolin on P-glycoprotein [54]. Another struc-
turally related compound, IBMX, currently used as a
nonspecific phosphodiesterase inhibitor, proved an effective
inhibitor of cGMP transport at 100 l
M
. The PKC inhi-
bitors, staurosporine and Ro 31–8220, inhibited the cGMP
uptake. Staurosporine completely inhibited the cGMP
uptake at 10 l
M
while at the same concentration Ro 31–
8220 inhibited by about 25%. The greater potency of
staurosporine is consistent with a recently proposed mech-
anism for its action: interaction with the ATP binding sites
of the transporter inhibiting energy-dependent drug efflux
activity [49].
The major characteristics of the cGMP transporter in

human erythrocytes, as described in the present study, are
inhibition by MK-571, glibenclamide, E
2
17bG, methotrex-
ate, DNP-SG and cAMP. This inhibitor profile matches
well with that for MRP4. At present, five distinguishing
features exist between human MRP4 and MRP5. First,
MRP4 is inhibited by MK-571 [39] while MRP5 is not [9].
Second, MRP4 transports methotrexate [39] but MRP5
does not confer resistance to this drug suggesting no
transport [55]. Third, human MRP4 transports 17bE
2
G[13]
but human MRP5 appears not to do so [9]. Fourthly,
cAMP and cGMP interact with MRP4 at similar concen-
trations [13] while the apparent K
D
for cAMP with MRP5 is
much higher than that for cGMP [9].
In summary, using inside-out membrane vesicles pre-
pared from human erythrocytes by a spontaneous, one-step
vesiculation process, a dominant low affinity component of
cGMP transport with K
d
value in the region of 50–80 l
M
has been identified. This transport is completely inhibitable
by MK-571, glibenclamide, clotrimazole, cAMP and
DNP-SG, consistent with the idea of a single transporter
being responsible. It is also markedly inhibited by metho-

trexate. All of these and cGMP block low affinity [
3
H]DNP-
SG transport in human erythrocytes and so the low affinity
transport of DNP-SG and the transport of cGMP may be
mediated by one and the same transporter. The character-
istics of transport indicate that MRP1, which mediates the
high-affinity transport of DNP-SG, is not the protein
responsible. The properties of the cGMP transport are
similar to those for MRP4.
Acknowledgements
We would like to thank Dr G. Kruh for the anti-MRP4 Ig, Dr G.
Scheffer and Dr R. J. Scheper for the M
5
I-1IgandDrM.Turnerfor
the MK-571.
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