Báo cáo khoa học: cGMP transport by vesicles from human and mouse erythrocytes potx

cGMP transport by vesicles from human and mouse
erythrocytes
Cornelia J. F. de Wolf
1
, Hiroaki Yamaguchi
1,
*, Ingrid van der Heijden
1
, Peter R. Wielinga
1,†
,
Stefanie L. Hundscheid
1,‡
, Nobuhito Ono
1,§
, George L. Scheffer
2
, Marcel de Haas
1
,
John D. Schuetz
3
, Jan Wijnholds
1,4
and Piet Borst
1
1 Department of Molecular Biology, the Netherlands Cancer Institute, Amsterdam, the Netherlands
2 Department of Pathology, Free University Medical Center, Amsterdam, the Netherlands
3 Department of Pharmaceutical Sciences, St Jude Children’s Research Hospital, Memphis, TN, USA
4 Netherlands Institute for Neurosciences, Royal Netherlands Academy of Arts and Sciences (KNAW), Amsterdam, the Netherlands
Three ATP-binding cassette (ABC) proteins, the multi-

drug resistance-associated proteins (MRPs), MRP4,
MRP5 and MRP8, now known as ABCC4, ABCC5,
and ABCC11, have been reported to transport cGMP
out of cells in an ATP-dependent manner [1–9]. The
physiologic significance of cGMP transport by these
transporters has remained unclear, however, and the
reported affinity of ABCC4 and ABCC5 for cGMP
Keywords
ABCC4; ABCG2; cGMP; multidrug
resistance; multidrug resistance protein
(MRP)
Correspondence
P. Borst, Department of Molecular Biology,
the Netherlands Cancer Institute, 1066 CX,
Plesmanlaan 121, Amsterdam, the Netherlands
Fax: +31 20 6691383
Tel: +31 20 5122880
E-mail:
Present address
*Department of Pharmaceutical Sciences,
Tohoku University Hospital, Sendai, Japan
†National Institute for Public Health and
Environment (RIVM), Microbiological
Laboratory for Health Protection (MGB),
Bilthoven, the Netherlands
‡Division of Diagnostic Oncology, the
Netherlands Cancer Institute, Amsterdam,
the Netherlands
§The 2nd Department of Internal Medicine,
Faculty of Medicine, Kagoshima University,

Kagoshima, Japan
(Received 13 September 2006, revised
20 October 2006, accepted 13 November
2006)
doi:10.1111/j.1742-4658.2006.05591.x
cGMP secretion from cells can be mediated by ATP-binding cassette
(ABC) transporters ABCC4, ABCC5, and ABCC11. Indirect evidence sug-
gests that ABCC4 and ABCC5 contribute to cGMP transport by erythro-
cytes. We have re-investigated the issue using erythrocytes from wild-type
and transporter knockout mice. Murine wild-type erythrocyte vesicles
transported cGMP with an apparent K
m
that was 100-fold higher than
their human counterparts, the apparent V
max
being similar. Whereas cGMP
transport into human vesicles was efficiently inhibited by the ABCC4-speci-
fic substrate prostaglandin E
1
, cGMP transport into mouse vesicles was
inhibited equally by Abcg2 and Abcc4 inhibitors ⁄ substrates. Similarly,
cGMP transport into vesicles from Abcc4
– ⁄ –
and Abcg2
– ⁄ –
mice was 42%
and 51% of that into wild-type mouse vesicles, respectively, whereas cGMP
transport into vesicles from Abcc4
– ⁄ –
⁄ Abcg2

– ⁄ –
mice was near background.
The knockout mice were used to show that Abcg2-mediated cGMP trans-
port occurred with lower affinity but higher V
max
than Abcc4-mediated
transport. Involvement of Abcg2 in cGMP transport by Abcc4
– ⁄ –
erythro-
cyte vesicles was supported by higher transport at pH 5.5 than at pH 7.4, a
characteristic of Abcg2-mediated transport. The relative contribution of
ABCC4 ⁄ Abcc4 and ABCG2 ⁄ Abcg2 in cGMP transport was confirmed with
a new inhibitor of ABCC4 transport, the protease inhibitor 4-(2-amino-
ethyl)benzenesulfonyl fluoride.
Abbreviations
ABC, ATP-binding cassette; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; Bcrp, murine breast cancer resistance protein; BCRP, human breast
cancer resistance protein; KO, knockout; MRP, multidrug resistance-associated protein; MTX, methotrexate; PG, prostaglandin.
FEBS Journal 274 (2007) 439–450 ª 2006 The Authors Journal compilation ª 2006 FEBS 439
differs widely depending on the investigator and
experimental method used [1,2,4,9].
The group of Sager characterized cGMP efflux from
human erythrocytes [10–15]. Subsequent studies with
various MRP inhibitors suggested that the major cGMP
transport system (low affinity) of erythrocytes has prop-
erties similar to those reported for ABCC4 [16–18].
However, Boadu & Sager [19] recently suggested that
ABCC5 is the major cGMP transporter in human eryth-
rocytes, based on their findings in ABCC5-depleted
human erythrocyte proteoliposomes. To further explore
this issue, we have turned to murine erythrocytes.

As knockout (KO) mice lacking specific ABC trans-
porters are available, it should be possible to unambigu-
ously determine the contribution of each transporter to
cGMP transport in these mice, rather than relying on
more or less specific inhibitors. Mice lacking Abcc4 have
been described [20]. Here we report the generation of
Abcc5
– ⁄ –
mice. Using these and other KO mice, we
found that at a substrate concentration of 1.8 lm
cGMP, about half of the cGMP transport by murine
erythrocyte vesicles is mediated by Abcg2 [murine breast
cancer resistance protein 1 (Bcrp1)], a transporter previ-
ously not known to transport nucleotides. The other
half is mediated by Abcc4. Abcc5 makes either a minor
or no contribution to cGMP transport. In contrast, our
results support the conclusion [16,17] that the bulk of
cGMP transport by vesicles from human erythrocytes is
attributable to ABCC4 and not to ABCC5 or ABCG2
[human breast cancer resistance protein (BCRP)].
Results
ABC transporters in mouse erythrocytes
To determine which of the ABC transporters that are
able to transport cGMP are present in the erythrocyte
membrane, we analyzed freshly isolated mouse erythro-
cytes by immunoblot, using Abcc1 and Abcg2 as
positive controls. Abcc4 and 5 were detected (Fig. 1).
Mice lack the ortholog of the human ABCC11 gene
[21]. Figure 1 also shows blots for erythrocytes of each
of the KO mice tested. Each KO mouse had indeed

lost the corresponding transporter, and the loss of one
transporter had not resulted in major secondary altera-
tions of the level of other transporters. However, we
note that we have not done serial dilutions of the pro-
tein loaded to determine more precisely whether minor
alterations (two-fold) do occur. For comparison,
Fig. 2 shows results obtained with human erythrocytes.
ABCC1, ABCC4, ABCC5 and ABCG2 were readily
detected (Fig. 2A), but ABCC11 was not (Fig. 2B).
Slight interindividual variations in ABCC1, ABCC4
and ABCG2 levels were observed between the human
volunteers, whereas larger variations in ABCC5 pro-
tein levels were seen. Although interindividual differ-
ences may be caused by variation in transporter
degradation between samples, the differences in
ABCC5 levels between individuals were repeatedly seen
in independent samples.
cGMP transport into membrane vesicles from
mouse erythrocytes
At a substrate concentration of 1.8 lm, the rate and
affinity of cGMP transport into mouse erythrocyte
vesicles (Fig. 3A–C) were much lower than reported
for human erythrocyte vesicles [16] and confirmed here
(K
m
¼ 132 ± 31 lm; Fig. 3D–F). This was due to the
low affinity of the murine transporters for cGMP, the
apparent K
m
being about 9 mm (9.0 ± 1.8 mm). This

is obviously a very rough estimate, as the maximal
concentration tested was 10 mm cGMP. The V
max
of
about 0.8 nmolÆ(mg protein)Æmin
)1
[0.76 ± 0.24 nmolÆ
(mg protein)Æmin
)1
] was comparable to that obtained
with human erythrocytes [0.39 ± 0.22 nmolÆ(mg protein)Æ
min
)1
).
Fig. 1. Levels of Abccs and Abcg2 in erythrocytes from WT and
KO mice. Western blot analysis of 10 lg of protein from mouse
erythrocyte vesicles. Each protein was detected as described in
Experimental procedures.
cGMP transport by erythrocytes C. J. F. de Wolf et al.
440 FEBS Journal 274 (2007) 439–450 ª 2006 The Authors Journal compilation ª 2006 FEBS
Inhibition of cGMP transport by MRP-specific
inhibitors and substrates
To test whether similar transport systems mediate
cGMP transport in human and mouse erythrocytes,
the effect of MRP inhibitors on cGMP transport was
assessed. The results are summarized in Table 1. The
sensitivity of cGMP transport into human erythrocyte
vesicles to MRP inhibitors was consistent with that
found in earlier studies [16,18]. In addition, we found
inhibition by low concentrations of prostaglandin

(PG) E
1
and PGE
2
. This is of interest, as these com-
pounds are relatively specific for ABCC4 and are not
detectably transported in vesicular transport experi-
ments by ABCC5 [22]. Less than 50% inhibition of
cGMP transport was obtained with the ABCG2
inhibitors Ko143 and GF120918. Significantly differ-
ent results were obtained with murine erythrocyte ves-
icles. On the one hand, PGE
1
and PGE
2
reduced
cGMP transport to only 57% and 59% of the con-
trol value, and the inhibitory effect of dipyridamole
and indomethacin was also less pronounced. On the
other hand, the Abcg2-specific inhibitor Ko143 inhib-
ited cGMP transport by the murine vesicles more
(52%) than cGMP transport by the human vesicles
(33%). These results raised the possibility that Abcg2
contributes to mouse erythrocyte cGMP transport (as
well as Abcc4), even though cGMP transport by
ABCG2 has not been reported before.
cGMP transport into erythrocyte membrane
vesicles from Abcc KO mice
Figure 4 shows the amount of cGMP transported after
30 min into vesicles from KO mice. Relative to wild-

type (WT) mice, the amounts obtained with Abcc4
– ⁄ –
,
Abcc1
– ⁄ –
⁄ Abcc4
– ⁄ –
, Abcc4
– ⁄ –
⁄ Abcc5
– ⁄ –
, Abcg2
– ⁄ –
and
Abcc4
– ⁄ –
⁄ Abcg2
– ⁄ –
mice were 42%, 42%, 39%, 51%
and 16%, respectively (P<0.01, as determined by
one-way anova). The differences in cGMP transport
between Abcc1
– ⁄ –
, Abcc5
– ⁄ –
and WT mice were not sig-
nificant.
Erythrocyte vesicles isolated from the Abcc4
– ⁄ –
⁄

Abcg2
– ⁄ –
mouse still transported cGMP at 16% of the
WT control level. As this value is close to background,
as reflected by the large standard deviation, its signifi-
cance is low. It may reflect a small contribution of
Abcc5 to cGMP transport, however, as the Abcc5
– ⁄ –
mouse also displayed a slight (not statistically signifi-
cant) reduction in cGMP transport. The borderline
transport remaining in the Abcc4
– ⁄ –
⁄ Abcg2
– ⁄ –
vesicles
shows that the inside-in vesicles present in our vesicle
preparations do not interfere with the cGMP transport
measurements.
Abcc4 and Abcg2 transport cGMP into mouse
erythrocyte vesicles
The results with inhibitors (Table 1) and KO mice
(Fig. 4A) indicated that Abcc4 and Abcg2 contribute
about equally to cGMP transport into mouse erythro-
cyte vesicles at the low substrate concentration used,
1.8 lm. We therefore made an attempt to determine
the kinetic constants for Abcc4- and Abcg2-mediated
cGMP transport using erythrocyte vesicles from the
KO mice, assuming that the remaining cGMP trans-
port in the Abcc4
– ⁄ –

mouse is due to Abcg2, and the
remaining transport in the Abcg2
– ⁄ –
mouse is due to to
Abcc4. The results are presented in Fig. 4B. At the
cGMP concentration routinely used for vesicular
uptake assays, 1.8 lm, Abcc4 and Abcg2 indeed con-
tributed equally to cGMP transport. However, at milli-
molar cGMP concentrations, Abcc4-specific cGMP
transport was saturable [V
max
¼ 0.20 ± 0.03 nmol
cGMPÆ(mg protein)Æmin
)1
], whereas saturation of
Abcg2-specific cGMP transport was not reached
[apparent V
max
about 1.4 nmol cGMPÆ(mg pro-
tein)Æmin
)1
]. Nonlinear regression analysis further
yielded an apparent K
m
of about 2.3 ± 0.9 mm for
cGMP transport by Abcc4, and an estimated apparent
K
m
>10mm for cGMP transport by Abcg2. This
shows that both murine transporters have a much

lower affinity for cGMP than human ABCC4.
AB
Fig. 2. Levels of ABCCs and ABCG2 in human erythrocytes.
(A) Western blot analysis of 10 lg of protein from human erythro-
cyte vesicles from five healthy volunteers (lanes 1–5). Each protein
was detected as described in Experimental procedures. (B) West-
ern blot analysis of 40 lg of protein from human erythrocyte vesi-
cles from three healthy volunteers (lanes 1–3). Lane 4: 10 lgof
protein from Sf9-hABCC11 cell lysate (positive control). Lane 5:
40 lg of protein from Sf9 WT cell lysate (negative control). Only
results obtained with monoclonal antibody M
8
II-16 are shown.
ABCC11 was detected as described in Experimental procedures.
h. ery ves, human erythrocyte vesicles.
C. J. F. de Wolf et al. cGMP transport by erythrocytes
FEBS Journal 274 (2007) 439–450 ª 2006 The Authors Journal compilation ª 2006 FEBS 441
The role of ABCG2/Abcg2 in cGMP transport into
human and mouse erythrocyte vesicles
To further characterize the contribution of Abcc4 and
ABCG2 ⁄ Abcg2 to erythrocyte cGMP transport, vesi-
cular uptake assays performed at physiologic pH were
compared with those done at low pH. Recently, it was
shown that ABCG2 transports methotrexate (MTX)
and resveratrol at a much higher rate at pH 6.0 than
at pH 7.4 [23]. In Fig. 5, we compare MTX (Fig. 5A)
and cGMP (Fig. 5B) transport into erythrocyte vesi-
cles at physiologic pH (pH 7.4) with transport at low
pH (pH 5.5). We confirmed the pH effect for murine
Abcg2 by demonstrating that MTX transport was

increased at pH 5.5 compared with pH 7.4 in vesicles
from WT and Abcc4
– ⁄ –
mice, whereas MTX transport
into vesicles derived from Abcg2
– ⁄ –
mice was not affec-
ted by low pH (Fig. 5A). Similarly, cGMP transport
into WT and Abcc4
– ⁄ –
mouse erythrocyte vesicles was
increased at low pH, whereas this pH effect was
absent from vesicles from Abcg2
– ⁄ –
mice (Fig. 5B).
However, whereas MTX transport into WT mouse
erythrocyte vesicles was increased 12-fold by low-pH
assay conditions, cGMP transport was increased only
two-fold. In contrast, low pH drastically decreased
transport of cGMP and MTX into human erythrocyte
vesicles. These results are compatible with a substan-
tial role for Abcg2 in cGMP transport by mouse
Fig. 3. Transport of cGMP into mouse and human erythrocyte vesicles. Erythrocyte membrane vesicles from five WT mice (A) or five
healthy volunteers (D) were incubated for the specified times at 37 °C with 1.8 l
M [
3
H]cGMP. Concentration-dependent transport of cGMP
into vesicles from four WT mice (B) or five healthy volunteers (E) was determined over a time span of 30 min. ATP-dependent transport
was calculated by subtracting the transport in the absence of ATP from that in the presence of ATP. Each point represents the mean ATP-
dependent cGMP transport ± SD. The background in the minus ATP control is illustrated in (C) and (F). Human erythrocyte vesicles 1–5

correspond to an individual subject, and are consistent throughout the figure (D, E). Erythrocyte vesicles isolated from a single mouse were
sufficient to perform a single experiment in triplicate. Therefore, mice 1–4 in (A) are not the same as mice 1–4 in (B).
cGMP transport by erythrocytes C. J. F. de Wolf et al.
442 FEBS Journal 274 (2007) 439–450 ª 2006 The Authors Journal compilation ª 2006 FEBS
erythrocytes and a negligible role for ABCG2 in
human erythrocytes.
4-(2-Aminoethyl) benzenesulfonyl fluoride
(AEBSF) inhibits Abcc4-specific cGMP transport
but not Abcg2-specific cGMP transport
AEBSF is an irreversible serine protease inhibitor [24]
that functions through acylation of serine residues in
the active site of the protease, resulting in sulfonate
ester formation [25]. As such, it is frequently included
in buffers and assay mixtures to prevent protein degra-
dation in plasma samples, in cell lysates, or in the
course of an enzymatic assay. However, AEBSF has
also been shown to bind to serine residues of other
proteins, and to a lesser extent also to tyrosine, lysine
and histidine residues, as well as the protein ⁄ peptide
N-terminus [26–29].
While optimizing the procedure for vesicle prepara-
tion, we observed an inhibitory effect of AEBSF on
acetylcholinesterase activity (reported also for the rela-
ted protease inhibitor phenylmethanesulfonyl fluoride
[30]) and, unexpectedly, also on the transport of
cGMP. Figure 6A shows the effect of three protease
inhibitors on cGMP transport into inside-out vesicles
prepared from human erythrocytes. In the concentra-
tion range recommended for the inhibition of protease
activity, leupeptin and aprotonin had a negligible effect

on vesicular uptake of cGMP. In contrast, complete
inhibition of cGMP transport into human erythrocyte
vesicles was already achieved at an AEBSF concentra-
tion of 5 mgÆmL
)1
(Fig. 6B). Preincubation at room
temperature of human inside-out erythrocyte vesicles
in transport assay buffer resulted in decreased cGMP
transport in incubations including 1 mg AEBSFÆmL
)1
but not in incubations lacking AEBSF (Fig. 6C). The
experiments were also performed with erythrocyte vesi-
cles from WT and KO mice to determine whether the
inhibition was transporter-specific or due to an overall
effect of AEBSF on the vesicles. cGMP transport into
erythrocyte inside-out vesicles from WT mice was
inhibited down to the level of transport observed for
vesicles from Abcc4
– ⁄ –
mice. In agreement with this,
cGMP uptake into vesicles from Abcc4
– ⁄ –
mice was
not affected by AEBSF, whereas AEBSF inhibited
cGMP uptake by vesicles from Abcg2
– ⁄ –
mice to the
same extent as observed for WT vesicles (Fig. 6D).
cGMP efflux from intact human erythrocytes
With intact HEK293 cells, we have previously reported

cGMP efflux mediated by ABCC4 or ABCC5 [4]. In an
attempt to show in vivo cGMP production and excretion
by human erythrocytes, we measured cGMP content as
well as cGMP efflux from freshly isolated and sodium
nitroprusside-stimulated erythrocytes, but we were
repeatedly unable to demonstrate the presence of cGMP
inside the erythrocytes, or of cGMP from the stimulated
Table 1. Effect of ABCC inhibitors and substrates on cGMP transport. Membrane vesicles from human and WT mouse erythrocytes were
coincubated for 30 min at 37 °C with 1.8 l
M [
3
H]cGMP and various established ABCC inhibitors ⁄ substrates. Each value was calculated by
subtracting ATP-dependent cGMP transport in the presence of inhibitor from that in the absence of inhibitor. Each value represents the
mean ± SD of duplicate measurements obtained from vesicles prepared from five individual mice or six human volunteers. Sample popula-
tions were tested for normality of distribution (Gaussian distribution). Student’s t-test, with Welch’s correction for unequal variance when
necessary, was performed to compare the degree of inhibition observed for each condition for mouse and human erythrocyte vesicles. The
Mann–Whitney test was performed when the sample size was too small (n ¼ 4) for an accurate estimation of sample distribution. NS, not
significant.
Inhibitor
Concentration
(l
M)
Erythrocyte vesicles
Mouse (n ¼ 5)
Transport
(% of control)
Human (n ¼ 6)
Transport
(% of control)
Student’s t-test

Mouse versus human
Dipyridamole 10 39.1 ± 6.6 26.1 ± 5.9 P ¼ 0.01
50 20.6 ± 12.3 5.0 ± 1.8 P<0.05
Indomethacin 10 62.2 ± 17.9 5.1 ± 1.0 P<0.01
50 42.9 ± 3.3
a
0.9 ± 1.3 P<0.05
MK571 5 35.2 ± 5.7
a
9.0 ± 0.9
a
P<0.05
PGE
1
20 57.4 ± 11.1 2.0 ± 1.0 P<0.001
PGE
2
20 59.2 ± 8.6 4.1 ± 1.8 P<0.001
Ko143 5 47.8 ± 18.9 67.1 ± 14.7 NS
GF120918 5 55.9 ± 19.4 58.5 ± 10.9 NS
a
Average of measurements from four individuals.
C. J. F. de Wolf et al. cGMP transport by erythrocytes
FEBS Journal 274 (2007) 439–450 ª 2006 The Authors Journal compilation ª 2006 FEBS 443
erythrocytes in the medium (results not shown). The
previously described HEK293 cells transfected with
ABCC4 cDNA [4] were included as a positive control,
and did secrete cGMP. It should be noted, however, that
the expression of MRP4 in the HEK293 cells is much
higher than the expression of MRP4 in erythrocytes;

5–10-fold as estimated from western blots.
Discussion
We have used murine erythrocytes to obtain more
insight into the nature of the cGMP transporters pre-
sent in the erythrocyte membrane. At low cGMP con-
centrations (1.8 lm), Abcc4 and Abcg2 contribute
equally to vesicular transport, as shown by the fact
that transport into vesicles from Abcc4
– ⁄ –
or Abcg2
– ⁄ –
mice is about half that into WT vesicles (Fig. 4A).
At higher cGMP concentrations, Abcg2 contributes
more, as its apparent V
max
is higher than that of
Abcc4; 1.4 versus 0.2 nmol cGMPÆ(mg protein)Æmin
)1
,
Fig. 4. Transport of cGMP into erythrocyte vesicles from WT and
KO mice. (A) Erythrocyte membrane vesicles from WT and KO
mice were incubated for 30 min at 37 °C with 1.8 l
M [
3
H]cGMP.
ATP-dependent transport of cGMP into vesicles from WT mice was
set to 100%. (B) Concentration-dependent transport of cGMP, 0.5–
10 m
M, into vesicles from WT (h), Abcc4
– ⁄ –

(.), Abcg2
– ⁄ –
(d) and
Abcc4
– ⁄ –
⁄ Abcg2
– ⁄ –
(s) mice was determined over a time span of
30 min. ATP-dependent transport was calculated by subtracting the
transport in the absence of ATP from that in the presence of ATP.
Each value represents the mean ± SD of duplicate measurements
from at least three individual mice.
Fig. 5. Effect of pH on MTX and cGMP transport into membrane
vesicles from humans and from WT and KO mice. (A) Effect of pH
on MTX transport. Erythrocyte membrane vesicles from humans
and WT and KO mice were incubated for 10 min at 37 °C with
1 l
M [
3
H]MTX at either pH 7.4 (j) or pH 5.5 (h). (B) Effect of pH
on cGMP transport. Erythrocyte membrane vesicles from WT and
KO mice were incubated for 30 min at 37 °C with 1.8 l
M
[
3
H]cGMP at either pH 7.4 (j) or pH 5.5 (h). For both panels, ATP-
dependent transport was calculated by subtracting the transport in
the absence of ATP from that in the presence of ATP. Substrate
transport into vesicles from WT mice at pH 7.4 was set to 100%.
The vesicle uptake buffer was 10 m

M Tris at either pH 7.4 or
pH 5.5. The final pH was verified by measurement with a pH
meter. Each value represents the mean ± SD of duplicate measure-
ments from three individuals ⁄ mice. For these experiments, erythro-
cyte vesicles from human individuals 1, 2 and 3 from Fig. 3 were
used.
cGMP transport by erythrocytes C. J. F. de Wolf et al.
444 FEBS Journal 274 (2007) 439–450 ª 2006 The Authors Journal compilation ª 2006 FEBS
respectively (Fig. 4B). The ability of Abcg2 to trans-
port cGMP has not been noted before. This is sup-
ported not only by the experiments with the Abcg2
– ⁄ –
erythrocyte vesicles, but also by the increased cGMP
transport at pH 5.5 (Fig. 5B), which is specific for
the Abcg2 fraction of cGMP transport. Increased
transport of MTX and resveratrol by human ABCG2
at acidic pH was first noted by Breedveld et al. [23],
but it is clear from Fig. 5A that it also applies to
murine Abcg2 and to the substrate cGMP (Fig. 5B),
although the pH effect on cGMP transport is less
pronounced than on MTX transport. Whether trans-
port of cGMP by Abcg2 has any physiologic signifi-
cance is doubtful, given the very low affinity of
Abcg2 for this substrate. The low rate of cGMP
transport by Abcg2 at substrate concentrations below
100 lm may also explain why this Abcg2 activity has
not been noted before. The ability of other ABC
transporters, such as ABCC4, ABCC5 and ABCC8,
to transport cyclic nucleotides is accompanied by the
ability to transport nucleotide analogs. Indeed, Wang

et al. [31,32] have reported that ABCG2 overexpres-
sion induces low-level resistance to some antiviral
nucleoside analogs, presumably through increased
excretion of the corresponding nucleotide analogs,
and we have recently found that Abcg2 confers high-
level resistance to the nucleoside analog cladribine
(unpublished results).
Our results for human erythrocyte vesicles confirm
and extend the conclusions of Klokouzas et al. [16]
and Wu et al. [18], in that cGMP transport by these
vesicles is attributable to ABCC4. We found > 95%
inhibition by PGE
1
and PGE
2
, at present the most
ABCC4-specific substrates known [22], and a complete
block of cGMP transport by the protease inhibitor
AEBSF, which seems to be relatively specific for
ABCC4, as we have not found inhibition by this com-
pound of ABCG2 ⁄ Abcg2 (Fig. 6). We note in passing
that the inhibition of ABCC4 by AEBSF is a compli-
cation that should be kept in mind, as protease inhib-
itor cocktails are often used routinely in vesicular
transport experiments.
AC
B
D
Fig. 6. Effect of AEBSF, aprotinin and leupeptin on cGMP transport into membrane vesicles from humans and from WT and KO mice.
(A) Effect of three different protease inhibitors on cGMP transport by human erythrocyte vesicles. Erythrocyte membrane vesicles were co-

incubated for 30 min at 37 °C with 1.8 l
M [
3
H]cGMP and the indicated concentration of either AEBSF, leupeptin or aprotinin. (B) Concentra-
tion-dependent effect of AEBSF on cGMP transport by human erythrocyte vesicles. Erythrocyte membrane vesicles were coincubated for
30 min at 37 °C with 1.8 l
M [
3
H]cGMP and AEBSF in the concentration range of 0.5–10 mg AEBSF per milliliter of incubation mix. (C) Effect
of preincubation of human erythrocyte vesicles with AEBSF on cGMP transport. Vesicles were preincubated at room temperature with (h)
or without (j) 1 mg of AEBSF per milliliter of incubation mix for either 0, 30 or 60 min. The length of preincubation time is shown on the
x-axis. Transport reactions were initiated by addition of 4 m
M ATP. (D) Concentration-dependent effect of AEBSF on cGMP transport by WT
and KO mouse erythrocyte vesicles. Erythrocyte membrane vesicles from WT (j), Abcc4
– ⁄ –
(j), Abcg2
– ⁄ –
(h) and Abcc4
– ⁄ –
⁄ Abcg2
– ⁄ –
(j)
mice were coincubated for 30 min at 37 °C with 1.8 l
M [
3
H]cGMP and 0, 0.1, 0.5 or 1 mg of AEBSF per milliliter of incubation mix. ATP-
dependent cGMP transport activity by vesicles from WT mice without addition of AEBSF were set to 100%; all other values are relative to
this value. All panels display the ATP-dependent transport of cGMP, which was calculated by subtracting the transport in the absence of
ATP from that in the presence of ATP. Each value represents the mean ± SD of duplicate measurements from three individuals ⁄ mice.
C. J. F. de Wolf et al. cGMP transport by erythrocytes

FEBS Journal 274 (2007) 439–450 ª 2006 The Authors Journal compilation ª 2006 FEBS 445
Although ABCG2 is present in human erythrocytes
(Fig. 2), it does not appear to significantly contribute
to cGMP transport, as there is no detectable transport
at pH 5.5 (Fig. 5B). The data in Fig. 5 indicate that
neither human ABCC4 nor murine Abcc4 transports
any cGMP at pH 5.5. Why the ABCG2-specific inhib-
itor Ko143 appears to inhibit cGMP transport into
human erythrocyte inside-out vesicles (Table 1) is
unclear. It seems likely that this is a nonspecific inhibi-
tory effect, like the inhibition by GF120918. Whether
the ABCC5 that is clearly present in human (Fig. 2)
and murine (Fig. 1) erythrocytes contributes at all to
cGMP transport is uncertain. There are no inhibitors
specific for ABCC5, and our results with the KO mice
(Fig. 4) are not unambiguous. Although the absence of
Abcc5 in the KO mice tends to lower the transport
rate somewhat, the effect is minimal and not statis-
tically significant. A substantial contribution of
ABCC5 ⁄ Abcc5 to erythrocyte cGMP transport, as
postulated by Boadu & Sager [19], is therefore ruled
out by our results. Boadu & Sager [19] measured
cGMP transport by protein fractions immunoprecipi-
tated from a detergent extract of erythrocytes and
reconstituted in proteoliposomes. In our opinion, the
authors provide no evidence that this approach can be
used as a quantitative assay for transport activity.
The low rate of cGMP transport by murine erythro-
cyte vesicles relative to their human counterparts is
clearly not due to differences in V

max
, but to the low
affinity of the murine transporters for cGMP, resulting
in minimal transport at the cGMP concentration
(1.8 lm) used in Fig. 3. Figure 4 shows that this low
affinity holds for both the Abcc4 and the Abcg2 com-
ponents of cGMP transport by murine erythrocytes.
What could be the physiologic role of ABCC4 activity
in erythrocytes? We have been unable to detect cGMP
in erythrocytes or cGMP efflux from erythrocytes after
stimulation, ruling out a role for ABCC4 in cGMP
transport in mature erythrocytes. It is possible that
ABCC4 is involved in secretion of cGMP from an eryth-
roid precursor cell, and that the ABCC4 in mature
erythrocytes is just a leftover, caused by the long half-
life of ABCCs [33]. Given the very low (mm) affinity of
murine Abcc4 for cGMP (Fig. 4B), it seems unlikely,
however, that cGMP transport is a normal function of
ABCC4 at all. Further studies with the Abcc4 and
Abcc5 KO mice now available should help to settle the
question of whether these transporters have any physio-
logic role as cyclic nucleotide transporters [34].
Mouse models are routinely used for the purpose of
drug resistance testing in cancer and antiviral research.
Erythrocytes may function as a carrier system in the
transport of endogenous compounds and xenobiotics,
such as the anticancer agents 6-mercaptopurine and
thioguanine, through the body. Active low-affinity,
high-capacity efflux of these compounds and their
metabolites from the erythrocyte by ABCC4 might

affect the bioavailability of these drugs [35]. However,
our finding that murine and human Abcc4 ⁄ ABCC4 and
Abcg2 ⁄ ABCG2 differ greatly in their affinity for cGMP
raises the question of whether this also holds for other
substrates, such as nucleoside analog drugs. Hence, we
are performing in vitro experiments to further examine
potential differences in substrate affinity between human
and murine variants of the ABCC ⁄ Abcc transporters.
Experimental procedures
Animals
Abcc4
– ⁄ –
[20], Abcc1
– ⁄ –
[36] and Abcg2
– ⁄ –
[37] mice were
generated previously. The Abcc5
– ⁄ –
mouse was generated
by J. Wijnholds through Abcc5 gene targeting. Briefly, a
sequenced 0.3 kb mouse Abcc5 cDNA fragment containing
sequences encoding the first ATP-binding domain of Abcc5
was used to screen an EMBL3 genomic 129 ⁄ Ola DNA
phage library. Four identical phage clones were character-
ized by Southern blotting, and exon–intron boundaries
were mapped. A targeting vector was constructed by assem-
bling a 4.1 kb SacI–EcoRV 5¢-Abcc5 genomic fragment, a
fragment containing a hygromycin resistance gene driven
by the mouse phosphoglycerate kinase promoter in reverse

orientation, and a 3.4 kb SmaI–StuI3¢ fragment of the
Abcc5 gene. Correct targeting deleted 1.5 kb of Abcc5
sequences containing exon 17 encoding amino acids 678–
745 of the first ATP-binding domain. Transfection of the
targeting construct into 129 ⁄ Ola-derived E14 embryonic
stem (ES) cells resulted in 10% homologous recombinants.
Targeted clones with the predicted replacement event were
identified by using probes 5¢ and 3¢ to the homology region.
Two of the ES cell clones with normal karyotype were
injected into mouse blastocysts, and both resulted in
chimeric mice that transmitted the Abcc5 mutant allele
through the germline of F1 offspring. The homozygous
mice were backcrossed to 100% Friend virus B-type (FVB)
genetic background. Double-KO mice, Abcc1
– ⁄ –
⁄ Abcc4
– ⁄ –
,
Abcc4
– ⁄ –
⁄ Abcc5
– ⁄ –
and Abcc4
– ⁄ –
⁄ Abcg2
– ⁄ –
, were generated
by crossbreeding of the single-KO mice. Male and
female Abcc1
– ⁄ –

, Abcc4
– ⁄ –
, Abcc5
– ⁄ –
, Abcc1 ⁄ 4
– ⁄ –
, Abcc4
– ⁄ –
⁄
Abcc5
– ⁄ –
, Abcg2
– ⁄ –
and Abcc4
– ⁄ –
⁄ Abcg2
– ⁄ –
mice and WT
mice were of comparable genetic background (FVB or
mixed Ola ⁄ B6 and FVB) and were killed between 9 and
14 weeks of age. Animals were kept in a temperature-con-
trolled environment with a 12 h light ⁄ 12 h dark cycle. They
received a standard diet and acidified water ad libitum.
Mice were housed and handled according to institutional
guidelines complying with Dutch legislation.
cGMP transport by erythrocytes C. J. F. de Wolf et al.
446 FEBS Journal 274 (2007) 439–450 ª 2006 The Authors Journal compilation ª 2006 FEBS
Blood sampling
Five milliliters of whole blood (heparin) was drawn from
healthy Caucasian volunteers by vein puncture. One milli-

liter of whole blood (heparin) was drawn from mice by
heart blood sampling under methoxyflurane anesthesia,
after which the mice were killed. Mouse handling and
experimental procedures were conducted in accordance with
institutional guidelines for animal care and use. All human
volunteers had given their consent for vein puncture.
cGMP efflux from intact cells
cGMP efflux from intact stimulated erythrocytes and
erythrocytic cGMP contents were measured with the direct
cGMP enzyme immunoassay (Assay Designs, Ann Arbor,
MI, USA) according to the manufacturer’s instructions.
Preparation of membrane vesicles from mouse
and human erythrocytes
Membrane vesicles from human and mouse erythrocytes
were prepared as previously described, with minor modifi-
cations [16]. Briefly, red blood cells were washed three times
with five volumes of isotonic medium (80 mm KCl, 70 mm
NaCl, 0.2 mm MgCl
2
,10mm Hepes, 0.1 mm EGTA,
pH 7.5). The buffy coat and top layer were removed after
each wash. The packed cells were lysed in 50 volumes of
ice-cold solution L (2 mm Hepes, 0.1 mm EGTA, pH 7.5)
and subsequently centrifuged at 20 000 g for 20 min at
4 °C. The supernatant was removed, and the pelleted ghosts
were resuspended in ice-cold solution L. This step was
repeated until most erythrocytes were lysed, as checked by
microscopy. The pellets were subsequently resuspended in
twice the packed red blood cell volume of solution L and
incubated at 37 °C for 30 min with occasional vortexing.

After incubation, the suspension was washed once with
solution L and twice with vesicle buffer (10 mm Tris ⁄ HCl,
pH 7.4). The final pellet was resuspended in vesicle buffer,
and the protein concentration was determined using the
Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). All
vesicles were prepared without protease inhibitors, unless
otherwise indicated. Membrane vesicles were frozen and
stored at ) 80 °C until use. To estimate the proportion of
inside-out vesicles, the activity of the ectoenzyme acetylcho-
linesterase was determined [11]. Routinely, 32–40% of vesi-
cles were inside-out, and there was no difference between
the inside-out ratios of vesicles from human or mouse
origin.
Vesicular transport assay
[8-
3
H]cGMP and [3¢,5¢,7-
3
H(N)]MTX (Moravek Biochemi-
cals, Brea, CA, USA) were used as substrates in vesicular
transport experiments. Substrate uptake into inside-out
erythrocyte vesicles was studied by use of the rapid filtra-
tion method as described previously [38]. Briefly, vesicles
containing 10 lg of protein were incubated with the indica-
ted concentration of substrate in a final volume of 25 lL
of vesicle buffer containing 10 mm MgCl
2
,10mm creatine
phosphate and creatine kinase (100 lgÆmL
)1

) (both from
Boehringer Mannheim, Almere, the Netherlands) in the
presence or absence of 4 mm ATP. Vesicular transport
assays were either performed at physiologic pH (pH 7.4) or
at pH 5.5. For the experiments at low pH, all reaction
components were prepared in 10 mm Tris (pH 5.5). The
pH of the final incubation mix was verified with a pH
meter. (We realize that the buffering capacity of this
pH 5.5 mix is very low; it was used to keep the conditions
of the transport experiment as similar as possible to the
conditions at pH 7.4.) At the indicated time, the reaction
was terminated by adding 2 mL of ice-cold vesicle buffer,
and the mixture was immediately filtered through a pure
cellulose ME25 (cGMP) or OE67 (MTX) filter (0.45 lm
pore size; Schleicher and Schuell, Dassel, Germany). The
filter was washed three times with 2 mL of ice-cold vesicle
buffer, and the radioactivity retained on the filter was
measured by liquid scintillation counting. The ATP-
dependent transport was calculated by subtracting the
transport in the absence of ATP from that in its presence.
Note that, initially, we determined ATP-dependent trans-
port by replacing ATP with 5¢-AMP; this gave the same
background as reactions performed in the absence of ATP.
cGMP was stable for 4 h at 37 °C, with intact cells trans-
porting cGMP into the medium, as measured with a valid-
ated HPLC method [4]. For inhibition studies, cGMP
uptake in the absence and presence of inhibitors was com-
pared. The MRP inhibitors MK571 (Biomol, Plymouth
Meeting, PA, USA), GF120918 (Glaxo Wellcome,
Research Triangle Park, NC, USA), Ko143 [39], PGE

1
and
PGE
2
(Sigma Aldrich, Zwindrecht, the Netherlands),
dipyridamole (Sigma Aldrich) and indomethacin (Sigma
Aldrich) were used. The inhibitory effect of the protease
inhibitors AEBSF, leupeptin and aprotinin (all from Roche
Applied Science, Indianapolis, IN, USA) on the vesicular
uptake of cGMP was determined in the concentration ran-
ges of 0–10 mg AEBSFÆmL
)1
, 0–5 lg leupeptinÆmL
)1
, and
0–2 lg aprotininÆmL
)1
. The vesicles were not preincubated
with inhibitors, the only exception being the experiment
shown in Fig. 6C. Kinetic parameters were calculated using
the equation V ¼ V
max
· S ⁄ (K
m
+ S), where V is the
transport rate [pmolÆ(mg protein)Æmin
)1
], S is the substrate
concentration in the buffer, K
m

is the Michaelis–Menten
constant, and V
max
is the extrapolated maximum velocity
[pmolÆ(mg protein)Æmin
)1
] at infinite S. The data were
fitted to the equation by nonlinear least-squares regression
analysis.
C. J. F. de Wolf et al. cGMP transport by erythrocytes
FEBS Journal 274 (2007) 439–450 ª 2006 The Authors Journal compilation ª 2006 FEBS 447
Generation of ABCC11 antibodies
Fusion genes consisting of the gene for the Escherichia coli
maltose-binding protein and fragments of human ABCC11
were constructed in the pMAL-c vector as previously des-
cribed [40]. The ABCC11 segment in the expression plasmid
encoded either amino acids 1–83 (FP M
8
I) or amino acids
455–526 (FP M
8
II). Production and purification of the
fusion proteins was performed as previously described [41].
Polyclonal rabbit anti-(human ABCC11) serum was
obtained from a rabbit immunized with FP M
8
I. For the
generation of monoclonal antibodies, a 12-week-old female
Wistar rat received approximately 30 lg of either FP M
8

I
or a 1 : 7 mix of FP M
8
II fusion protein and a synthetic
ABCC11 peptide (amino acids 475–526) per injection.
Three booster injections were given. Cells obtained from
draining lymph nodes and the spleen were fused with Sp
2
0
mouse myeloma cells as previously described [42,43]. Rat
monoclonal antibodies M
8
I-74 and M
8
II-16 were selected
by screening hybridoma supernatants on ELISA plates coa-
ted with FP M
8
II and, as a control, on plates coated with
irrelevant fusion protein. Antibody binding was detected
using horseradish peroxidase-labeled rabbit anti-rat serum
(1 : 500; Dako, Glostrup, Denmark) and 5-amino-
2-hydroxybenzoic acid (Merck, Darmstadt, Germany) with
0.02% H
2
O
2
as a chromogen. Human recombinant
ABCC11 expressed in Sf9 insect cells was specifically detec-
ted with both the rabbit polyclonal anti-(human ABCC11)

serum and the rat monoclonal antibodies M
8
I-74 and M
8
II-
16 (Fig. 2B and results not shown).
Western blot analysis
Membrane vesicles (10 lg of protein) were fractionated on
a denaturing 7.5% polyacrylamide gel and transferred onto
a nitrocellulose membrane. Forty micrograms of vesicular
protein was loaded onto a polyacrylamide gel for the detec-
tion of ABCC11. Equal loading and transfer of protein was
routinely checked by Ponceau S staining of the nitrocellu-
lose membrane. After blocking for 1 h in NaCl ⁄ P
i
contain-
ing 1% nonfat dry milk, 1% BSA, and 0.05% Tween-20,
the membrane was incubated for 1 h at room temperature
with the first antibody. ABCC (Abcc) 1, 4 and 5 and
ABCG2 (Abcg2) were detected with the monoclonal anti-
bodies ABCC-r1 [44] (1 : 1000), M
4
I-10 [20] (1 : 500), NKI-
12C5 [45] (1 : 1) and BXP-53 [37] (1 : 400), respectively.
For the detection of ABCC11, the polyclonal (1 : 1) and
monoclonal (1 : 5) ABCC11 antibodies described in the
previous section were used. As secondary antibody, horse-
radish peroxidase-conjugated rabbit anti-(rat IgG) or swine
anti-(rabbit IgG) was used at a dilution of 1 : 1000 (Dako).
Enhanced chemiluminescence was used for detection by

incubating the membrane for 1 min with freshly mixed
1.25 mm 3-aminophtalhydrazide, 0.2 mm p-coumaric acid,
and 0.01% v ⁄ vH
2
O
2
in 0.1 m Tris (pH 8.5).
Acknowledgements
We thank A. Schinkel (Netherlands Cancer Institute)
for providing us with the Abcg2
– ⁄ –
mouse, and K. van
de Wetering of our group for the other mice. This
research was supported by grants from the Uehara
Memorial Foundation to H. Yamaguchi, the Dutch
Cancer Society to P. Borst (NKI 98-1794, and NKI
2001-2473) and J. Wijnholds (NKI 2001-2473), and NIH
research grants GM60904, ES058571, and CA23099,
Cancer Center Support Grant P30 CA21745, and a
grant from the American Lebanese Syrian Associated
Charities (ALSAC) to J. Schuetz. A major part of this
work was presented at the FEBS special meeting on
ABC proteins (Innsbruck, Austria, 4–10 March 2006).
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