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: p.borst@nki.nl 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). References 1 Jedlitschky G, Burchell B & Keppler D (2000) The mul- tidrug resistance protein 5 functions as an ATP-depen- dent export pump for cyclic nucleotides. J Biol Chem 275, 30069–33007. 2 Chen ZS, Lee K & Kruh GD (2001) Transport of cyclic nucleotides and estradiol 17-beta-D-glucuronide by mul- tidrug resistance protein 4. 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Erythrocyte membrane vesicles from humans and. 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