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

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.

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,

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

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

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

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

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
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

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

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

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
[

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

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

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

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

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

,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
– 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

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/

(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

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

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

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

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

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

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

)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

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

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

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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