Eur J Biochem 270, 3696–3708 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03753.x 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 possible 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 lM This transport is reduced by MRP inhibitors and substrates including MK-571, methotrexate, estradiol 17-b-D-glucuronide, and S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL-3 that inhibits high-affinity transport of DNP-SG by MRP1 It is concluded that the cGMP exporter is distinct from MRP1 and has properties similar to those reported for MRP4 Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes Active transport of the cyclic nucleotide cGMP across human erythrocyte membranes can be demonstrated using intact cells [1] or inside-out membrane vesicle preparations [2–4] In the studies using inside-out membrane vesicles, the active uptake of cGMP was found to be saturable with two components, one of high-affinity (Km 2–5 lM) [2,5] and another of low-affinity (Km 170 ± 50 lM) [5] Two components have also been described for the transport of another organic anion, a glutathione conjugate S(2,4dinitrophenyl)glutathione (DNP-SG), in human erythrocytes [6,7] Two members of the multidrug resistance-associated protein (MRP) transporter family, MRP1 and MRP5 have been detected previously in human erythrocyte membranes [8,9], and transport by MRP1 has been conclusively shown to account for the high-affinity component of DNP-SG transport [10–12] MRP4 and MRP5 have been shown to transport the cyclic nucleotides, cAMP and cGMP [9,13,14] and it has been suggested that MRP5 mediates the highaffinity 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 lowaffinity 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 insideout 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 lM This transport is reduced by a range of inhibitors and substrates for MRPs including MK-571, methotrexate, E217bG, and DNP-SG and also by glibenclamide and frusemide We show that this cGMP exporter is distinct from MRP1 and has characteristics similar to those reported for MRP4 The evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes Correspondence to S B Hladky, Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, UK Fax: + 44 1223 334040, Tel.: + 44 1223 334019, E-mail: sbh1@cam.ac.uk Abbreviations: ATP-c-S, adenosine 5¢-O-(3-thiotriphosphate); DNP-SG, S(2,4-dinitrophenyl)glutathione; E217bG, estradiol 17-bD-glucuronide; GSH, reduced glutathione; HRP, horseradish peroxidase; IBMX, isobutylmethylxanthine; MK-571, (3-([[3-(2-[7-chloro2-quinolinyl]ethenyl)phenyl}-{(3-(dimethylamino-3-oxopropyl)thio}-methyl]thio) propanoic acid; MRP, multidrug resistance-associated protein; PNGase F, peptide N-glycosidase F; Ro, 31–8220 bisindolylmaleimide; SITS, 4-acetamido-4¢-isothiocyano-2,2¢-disulfonic stilbene IX methanesulfonate  Present address: Laboratory of Cell Biology, National Cancer Institute, Building 37, Room 1B17, 37 Convent Drive, Bethesda, MD 20892, USA Note: web page available at http://www.phar.cam.ac.uk (Received 20 May 2003, revised 14 July 2003, accepted 15 July 2003) Keywords: multidrug resistance-associated protein; guanosine cyclic mono-phosphate transport; glutathione-conjugate transport; human erythrocytes; membrane vesicles Ó FEBS 2003 cGMP transport in human erythrocytes (Eur J Biochem 270) 3697 Experimental procedures Chemicals [8-3H]cGMP (specific activity 13.9 CiỈmmol)1) was obtained from Amersham Biosciences, [glycine-2-3H]GSH (specific activity 40 CiỈmmol)1) and [3H]glibenclamide (specific activity 44.7 CiỈmmol)1) were obtained from New England Nuclear, respectively M5I-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 M5I-1 and antiMRP4 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-chloro2,4-dinitrobenzene, clotrimazole, creatine kinase, creatine phosphokinase, daunorubicin, dideoxyforskolin, isobutylmethylxanthine (IBMX), doxorubicin, estradiol 17b-D-glucuronide, forskolin, glibenclamide, glutathione (reduced form, GSH), glutathione S-transferase, guanosine 3¢,5¢-cyclic monophosphate (cGMP), imidazole, indomethacin, leupeptin, lithocholic acid 3-sulphate, methotrexate, pepstatin A, probenecid, taurocholic acid, tetraethylammonium chloride, Triton X-100, Tween 20, verapamil and vincristine were all obtained from Sigma Chemicals Calcein was purchased from Molecular Probes Staurosporine and bisindolylmaleimide IX methanesulfonate (Ro 31–8220) were obtained from Calbiochem (3-([[3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-(dimethylamino-3-oxopropyl)-thio}-methyl]thio) propanoic acid, MK-571, was a generous gift of M Turner (Merck-Frosst Center for Therapeutic Research, Quebec, Canada) Peptide N-glycosidase F (PNGase F) was purchased from Promega Drugs were prepared in 10 mM Tris/HCl (pH 7.4) for GSH, ATP, ATP-c-S, cGMP, taurocholic acid, imidazole, vincristine and MK-571 or in 66% dimethyl sulfoxide/34% water for glibenclamide, SITS, methotrexate, verapamil, indomethacin, E217bG and clotrimazole The final concentration of the dimethyl sulfoxide did not exceed 0.5% in each experiment GSH stock solutions (adjusted to pH 7.4) were freshly prepared on the day of each experiment [3H]DNP-SG was synthesized enzymatically as previously described [12,20] The purity of the 3H-labelled DNPSG was determined by thin-layer chromatography on silica gel plates [(0.25 · 40 · 80) mm, AlugramÒSIL G/UV254, Macherey-Nagel, Germany] using n-propanol:water (7 : 3, v/v) as solvent [21] Cell lines COR-L23/R and MOR/R are MRP1-overexpressing, multidrug-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 mM), penicillin (100 ImL)1) and streptomycin (100 lgỈmL)1) (complete RPMI-1640) in a 5% CO2 humidified incubator at 37 °C The L23/R and MOR/R sublines were maintained in the presence of 0.2 lgỈmL)1 and 0.4 lgỈmL)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 confluent RNA protection assay of the doxorubicin-resistant COR-L23/R and MOR/R cells [24] shows that these cells: not express P-glycoprotein; over-express MRP1 when compared to the doxorubicin-sensitive controls; and express MRP4 at a low level MRP5 is expressed at very low level in COR-L23 cells but at high level in MOR cells Preparation of inside-out human lung tumour cell membrane vesicles Membrane vesicles from human lung tumour cells were prepared according to a method described previously [25] in the presence of protease inhibitors (5 lgỈmL)1 leupeptin, lgỈmL)1 aprotinin, 80 ngỈmL)1 pepstatin A) Briefly, cells were lysed in ice-cold hypotonic buffer (1 mM Tris/HCl, pH 7.4) for 30 at °C Following centrifugation at 100 000 g for 30 at °C, the resulting pellet was homogenized vigorously with a Teflon hand homogenizer in buffer containing 10 mM Tris/HCl, 250 mM sucrose, and protease inhibitors, layered over 38% (w/v) sucrose in 10 mM Tris/HCl and centrifuged at 100 000 g for 30 at °C The membranous material in the layer at the interface with the sucrose was collected, washed and centrifuged at 100 000 g for 30 at °C The resulting pellet was re-suspended in transport buffer (10 mM Tris/HCl, 250 mM 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 vesiculation process as previously described [26–28] with minor modifications Briefly, red blood cells were washed three times with vols of isotonic medium (80 mM KCl; 70 mM NaCl; 0.2 mM MgCl2; 10 mM Hepes; 0.1 mM EGTA, pH 7.5) Higher concentrations of EGTA (0.5–3 mM) and high pH (8.5) interfere with the vesiculation process [26] The buffy coat and topmost cell layer were removed after each wash The packed cells were then lysed by addition to 90 vols of ice-cold solution L (2 mM Hepes and 0.1 mM EGTA, pH 7.5) and subsequently centrifuged at 40 000 g for 20 at °C The supernatant was removed and the pelleted ghosts were re-suspended in ice-cold solution L This step was repeated twice After the last wash, the pellets were re-suspended by addition of half the original packed cell volume of cold solution L and incubated at 37 °C for 30 resulting in spontaneous formation of spectrinactin-free vesicles [28] After incubation, the suspension was washed with solution L and the resulting pellet resuspended in 10 mM Tris/HCl (pH 7.4) The protein concentrations of the vesicle samples were determined using the BCA (bicinchoninic acid) protein assay (Pierce) Membrane vesicles were frozen and stored at )80 °C until use Ó FEBS 2003 3698 A Klokouzas et al (Eur J Biochem 270) Measurement of membrane vesicle sidedness The proportion of inside-out vesicles in the membrane preparations was assessed by determining the accessibility of the ectoenzyme acetylcholinesterase, and the endoenzyme glyceraldehyde-3-phosphate dehydrogenase to their substrates 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 mM Tris/HCl (pH 7.4) for the assays involving membrane vesicles prepared from human erythrocytes 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 mM ATP, 10 mM MgCl2, 10 mM creatine phosphate, 100 lgỈmL)1 creatine kinase, 10 mM Tris/HCl (pH 7.4) and 3.3 lM [3H]cGMP or lM [3H]DNP-SG or 254 lM [3H]DNP-SG Aliquots (20 lL) were taken from the mixture after 15 in the case of cGMP uptake, after 30 with lM [3H]DNP-SG uptake, and after 45 with 254 lM [3H]DNP-SG uptake, diluted in mL of ice cold stop solution (10 mM 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 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 mM creatine phosphate, 100 lgỈml)1 creatine kinase) was present 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 mM Tris/HCl (pH, 7.4), dimethyl sulphoxide or ethanol, with the latter two solvents at a final concentration < 0.5% v/v], identical concentrations of the vehicle being used in control samples Curve fitting and statistics Data are reported as mean ± s.e.m Estimates of maximum uptake rates and apparent dissociation constants were obtained by least squares fits to the data using either the solver in Microsoft EXCELÒ or KALEIDAGRAPHÒ (Synergy Software, Reading, PA, USA) Measurements of uptake vs substrate concentration were fitted assuming that transport occurs as the sum of two processes each described by a Hill equation [32]: U¼ Umax cn ; K n ỵ cn D n 1ị where c is the concentration, Umax the maximum uptake, Kd the apparent dissociation constant and n is the Hill coefficient, here expected to lie between and The data were fitted to minimize the sum of squared proportional deviations, X Ui;observed Ui;fitted =Ui;fitted 2ị SSE ẳ i fits to inhibition curves were to equations of the form: Uẳ U0 Unoninh ị ICni 50 ỵ Unoninh ; ICni ỵ Ini 50 ẳ ni ẳ ð3Þ where I is the concentration of the inhibitor, IC50 is the inhibitor concentration producing 50% inhibition of the inhibitable component, U0 is the uptake in the absence of the inhibitor, Unoninh is the uptake which cannot be inhibited and ni is the Hill coefficient for the inhibitor For simple competition, ni ¼ Unless stated otherwise, KaleidaGraphỊ was used to fit the data using the variances at each concentration, ri, as the weights: X ÀÀ Á Á2 SSE ¼ v2 ¼ Ui;observed À Ui;fitted =ri ð4Þ i fits with different numbers of fitting parameters were compared using an F-test on the ratio of the variance associated with the reduction in degrees of freedom to the variance of the fit with the smaller number of degrees of freedom [33], VR ẳ SSE2 SSE1 ị=d.f.2 d.f.1 ị SSE1 =d.f.1 ð5Þ where d.f is the number of degrees of freedom (¼ number of data points ) number of fitting parameters) The improvement in fit is labelled ÔsignificantÕ if the probability from the F-test is less than 0.05 Fits with the same number of degrees of freedom were compared with each other using the likelihood ratio:   DSSE LR ¼ exp À ð6Þ 2r2 where DSSE is the difference in SSE for the two fits and the noise variance was estimated as r2 ¼ MinSSE/d.f with MinSSE equal to the value of SSE for the best fit SDS/PAGE and Western blotting Membrane vesicle or crude lysate proteins (5–40 lg) were separated through 7.5% (w/v) polyacrylamide and subsequently transferred onto Hybond ECL nitrocellulose membranes (Amersham Biosciences) Each membrane was then incubated in blocking buffer [5% (w/v) milk powder in 0.1% NaCl/Pi/Tris/Tween (25 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20)] overnight at °C prior to the addition of the primary Ig (M5I-1, : 40 dilution; Ó FEBS 2003 cGMP transport in human erythrocytes (Eur J Biochem 270) 3699 anti-MRP4, : 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 M5I-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 in the presence of 0.5% SDS and 1% b-mercaptoethanol, followed by incubation at 37 °C for h in the presence of 50 mM 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 (GlcNAc) and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins [34] PNGase F hydrolyzes nearly all types of N-glycan chains from glycopeptides/proteins Results ATP-dependent uptake of cGMP into human erythrocyte membrane vesicles The rate of ATP-dependent uptake of 3.3 lM [3H]cGMP at 37 °C into inside-out erythrocyte membrane vesicles was approximately constant for more than 30 at about 10 pmolỈmg)1Ỉmin)1 (Fig 1A) Uptake of [3H]cGMP in the absence of ATP but in the presence of the nonhydrolysable ATP analogue, ATP-c-S, was less than 5% of the uptake in the presence of ATP The amount of [3H]cGMP taken up by these vesicles was approximately twofold lower (44 ± 3%, mean ± SEM, n ¼ 4) when measured with NaCl/Pi (140 mM NaCl; mM KCl; 10 mM Na2HPO4; and 1.8 mM KH2PO4, pH 7.4) than with the usual low osmolality 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[3H]cGMP was determined at cGMP concentrations in the range 0.5–300 lM (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 and Table The data imply that there is a large (Umax > 300 pmolỈmg)1Ỉmin)1), weakly cooperative (n % 1.1–1.4) low affinity component with dissociation constant, Kd2, in the range 50–85 lM and suggest that there may also be a second, much smaller high affinity component with Kd1, in the range 0.5–2.5 lM However, this latter component, which may correspond to the uptake observed previously [2,5], contributes less than 20% of the uptake even for a low concentration, 3.3 lM, of cGMP ATP-dependent uptake of DNP-SG into inside-out, human erythrocyte vesicles When inside-out erythrocyte membrane vesicles were incubated at 37 °C with lM [3H]DNP-SG, the uptake in the presence of mM ATP increased linearly in time for at least Fig ATP-dependent uptake of cGMP into inside-out membrane vesicles prepared from human erythrocytes (Top) Uptake of 3.3 lM [3H]cGMP was measured in the presence of mM ATP or the nonhydrolysable 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 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 coefficient ¼ 1.09, and the solid curve for two components, each described by a Hill equation with Hill coefficients of for the high affinity, low capacity component and 1.3 for the low affinity, high capacity component 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 3700 A Klokouzas et al (Eur J Biochem 270) Table Fitting parameters for the uptake of [3H]cGMP into one-step, inside out erythrocyte membrane vesicles shown in Fig The maximum uptake rates, Umax1 and Umax2, the dissociation constants, Kd1 and Kd2, and the Hill coefficients, n1 and n2 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 relative to the column immediately to the left The fit obtained with the constraints n1 ¼ and n2 ¼ is not shown as the fitted value of Umax1 was 0.000 These data imply (F-test on the variance ratio, P ¼ 0.0004) that the low affinity component shows cooperativity, n2 > 1, and are consistent with the presence of a high affinity component (F-test, P ¼ 0.002), but not specify its characteristics Constraints Fitted constant Umax1/pmolỈmg)1Ỉ min)1 Kd1/lM n1 Umax2/pmolặmg)1ặ min)1 Kd2/lM n2 SSE VR P Umax1 ẳ n2 ¼ Umax1 ¼ n1 ¼ n1 < ¼ 0 7.1 2.7 – – 551 – – 389 2.35 293 0.674 304 150 1.00 0.543 – 82 1.09 0.353 15.7 0.0004 50 1.40 0.223 7.9 0.002 52 1.32 0.208 1.8 0.187 60 while uptake when ATP was replaced by ATP-c-S was almost negligible [12] ATP-dependent uptake of [3H]DNP-SG in human erythrocyte vesicles was determined over a broad concentration range (0.44–1000 lM) (Fig 2) As for cGMP, the data for DNP-SG were analyzed using a two component Hill equation The results of fits with several different restrictive assumptions are shown in Table The quantitative fitting (variance ratio test) confirms, as is obvious by eye, that the transport occurs via at least two components To explore the range of acceptable values of the Hill coefficient for the low-affinity component, n2, least squares fits were obtained for specified values of n2 (Table 2) Acceptable fits (likelihood ratio ¼ 0.05 compared to the best fit) were obtained for values of n2 between and 1.48 Over the range from to 1.4, the low-affinity dissociation constant decreases from 82 to 65 lM while that for the high-affinity component varies from 0.5 to lM In agreement with previous work [10,11] the high affinity component of DNP-SG transport in these vesicles is most likely mediated by MRP1 [12] Strong evidence in support of this comes from the observation that the uptake rate of lM 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 DNPSG concentration was increased to 254 lM The inhibition by QCRL-3 was then only 40 ± 5% (n ¼ 6) This result and the complete inhibition observed at low DNPSG concentrations suggests that there is some low Fig Rate of ATP-dependent uptake of [3H]DNP-SG into inside-out erythrocyte membrane vesicles The dotted curve is drawn for a single simple saturable component of uptake, the solid curve for two simple saturable components The dashed and dash-dot curves are drawn for two components each obeying a Hill equation with the constraints that n2 ¼ 1.4 or n2 ¼ 2, respectively The data are plotted directly (Top) and as a Haynes–Wolfe plot (Bottom) The fitted constants are described in Table These data are not consistent with a single-component of uptake, but cannot unambiguously determine the properties of two components when provision is made for the possibility that more than one substrate molecule may interact with the transporter at a time 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 lM This requirement is consistent with the uptake measurements provided n2 ¼ 1.4 All the data are consistent with high affinity transport via MRP1 (Kd ẳ lM, Umax ẳ 20 pmolặmg)1ặ min)1) and low affinity, weakly cooperative transport via a second transporter (Kd ¼ 65 lM, Umax ¼ 196 pmolỈ mg)1Ỉmin)1 and n2 ¼ 1.4) Interrelations between cGMP and DNP-SG uptake into human erythrocyte membrane vesicles To explore the relations between cGMP and DNP-SG transport, the ability of each to inhibit transport of the other was investigated ATP-dependent uptake of lM DNP-SG was not affected by the presence of cGMP at concentrations up to 500 lM (Fig 3A) suggesting that the high affinity DNP-SG transporter, MRP1, does not transport cGMP Ó FEBS 2003 cGMP transport in human erythrocytes (Eur J Biochem 270) 3701 Table Fitting parameters for uptake of [3H]DNP-SG into one-step, inside out erythrocyte membrane vesicles The maximum uptake rates, Umax1 and Umax2, the dissociation constants, Kd1 and Kd2, and the Hill coefficients, n1 and n2 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 n1 > ¼ 1, n2 > ¼ using the likelihood ratio (Eqn 6) These data are consistent with any value of n2 between and 1.48 (LR ¼ 0.05) Constraints Fitting constant Umax1/pmolỈmg)1Ỉmin)1 Kd1/lM n1 Umax2/pmolỈmg)1Ỉmin)1 Kd2/lM n2 AF CF SSE VR P LR Umax1 ¼ 0; n2 > ¼ n1 > ¼ 1; n2 > ¼ n1 > ¼ 1; n2 > ¼ 1.2 n1 > ¼ 1; n2 > ¼ 1.4 n1 > ¼ 1; n2 > ¼ – – 197 36 1.00 0.643 0.110 2.155 6.4 0.52 1.00 235 82 1.00 0.709 0.108 0.476 25.9 · 10)7 13.5 1.32 1.00 212 71.554 1.20 0.703 0.108 0.500 19.5 1.99 1.00 196 65 1.40 0.702 0.108 0.529 32.0 3.62 1.00 171 56 2.00 0.705 0.109 0.611 0.088 0.002 This finding was further supported by the observation that the MRP1-specific Ig, QCRL-3, produced negligible inhibition of the ATP-dependent uptake of cGMP (uptake rate for 3.3 lM cGMP with 10 lgỈmL)1 QCRL-3 was 98 ± 5% of control, n ¼ 3) On the other hand, ATP-dependent uptake of DNP-SG at high concentrations was inhibited by cGMP (Fig 3B) The fitted curve for uptake at 254 lM DNP-SG has two components: a noninhibitable uptake (30 min) of 2943 ± 115 pmolỈmg)1 and a component of 4110 ± 144 pmolỈmg)1 inhibitable by cGMP with an IC50 of 133 ± 18 lM These components are plausibly attributed to the high and lowaffinity 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 lM [3H]cGMP in inside-out membrane vesicles was measured in the presence of DNP-SG in the range of 0.5– 800 lM (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 IC50 82 ± lM and a Hill coefficient of 1.25 ± 0.02 lM Effect of MRP inhibitors, substrates, and modulators on cGMP uptake into human erythrocyte membrane vesicles: A number of compounds that are known to interact with one or more MRPs were tested for their ability to inhibit cGMP transport (see Fig and section below entitled ÔcGMP transport is inhibited by anion transport inhibitors, PKC inhibitors and IBMXÕ) MK-571, a leukotriene D4 (LTD4) receptor antagonist, which has been shown to inhibit transport by MRP1 [36,37], MRP2 and MRP3 [38] and MRP4- [39] but not MRP5-mediated cGMP transport [9] completely inhibited the [3H]cGMP uptake in vesicles with an IC50 value of 0.38 ± 0.01 lM (Fig 4A) suggesting that this transport is not mediated by MRP5 cAMP (Fig 4B) which is transported by MRP4 and MRP5 [9,13], 0.34 inhibited with IC50 ¼ 296 ± 26 lM The ratio of this constant to the apparent dissociation constant for cGMP, 50–80 lM (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 E217bG, 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 concentrations sufficiently high (> 200 lM, 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 mM, neither enhanced nor inhibited cGMP uptake (Table 3) This contrasts with the effect of 1–5 mM GSH to stimulate uptake of DNP-SG in human erythrocyte vesicles [12] but is consistent with the lack of effect of GSH on MRP4 and MRP5 mediated transport in transfected HEK293 cells [17] The cationic vinca alkaloid, vincristine, and the organic anion, calcein, which are established MRP1 substrates, were also tested for their ability to inhibit cGMP uptake Calcein inhibited cGMP uptake (about 25% inhibition at 100 lM and about 60% at 300 lM) but vincristine itself had no effect even at 200 lM (Table 3) It is known that several cationic MRP1 substrates, including vincristine, require GSH for their transport and that vincristine inhibits the high affinity DNP-SG transport in the presence but not in the absence of GSH [12,45,46] Thus the effect of vincristine on the cGMP uptake was also tested in the presence of Ó FEBS 2003 3702 A Klokouzas et al (Eur J Biochem 270) mM GSH but no inhibition was observed Given that MRP1-mediated transport of calcein in whole cells can be modulated by the level of GSH present [47], the effect of calcein on cGMP uptake into erythrocyte membrane vesicles was also tested in the presence of mM GSH but no additional inhibition was observed (Table 3) These results are all consistent with cGMP transport via MRP4 while the strong inhibition produced by MK-571 and the relative potency of cAMP appear to be incompatible with transport via MRP5 CGMP transport is inhibited by anion transport inhibitors, PKC inhibitors and IBMX Because substrates for MRPs are often organic anions, inhibitors that block ion transport were tested (Table 4) The anion transport inhibitors frusemide, niflumic acid, phloridzin, SITS and probenecid all reduced the rate of cGMP uptake (Table 4) By contrast the potassium channel blockers, 4-aminopyridine, tetraethylammonium chloride and CsCl, had no observable effect though BaCl2 did (Table 4) Verapamil, a calcium channel blocker, an inhibitor of P-glycoprotein, and a general though weak inhibitor of MRPs in vesicular drug uptake studies, reduced cGMP transport in the presence or absence of mM GSH Two protein kinase C inhibitors, staurosporine and Ro 31–8220 [48], were also tested for their ability to block cGMP transport in human erythrocytes Staurosporine has recently been shown to bind directly to several ABC transporters [49] in addition to preventing phosphorylation of these transporters in intact cells [50] Staurosporine at 10 lM completely inhibited the cGMP uptake while Ro 31–8220 at 10 lM showed only weak inhibition Forskolin, an activator of adenylyl cyclase, inhibited while its inactive analogue, 1,9-dideoxyforskolin, had no effect at the same concentration 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 Fig Effect of cGMP on the DNP-SG transport in human erythrocytes and vice versa (Top) ATP-dependent uptake of lM [3H]DNPSG (30 at 37 °C) was not affected by cGMP at concentrations up to 500 lM (Middle) ATP-dependent uptake of 254 lM [3H]DNP-SG (30 at 37 °C) was partially inhibited by cGMP The fitted curve corresponds to two components: a noninhibitable component of 2943 ± 115 pmolỈmg)1 and a component of 4110 ± 144 pmolỈmg)1 inhibited by cGMP with an IC50 of 133 ± 18 lM (mean ± SEM) (Bottom) ATP-dependent uptake of 3.3 lM [3H]cGMP (15 at 37 °C) was inhibited by DNP-SG as a single component described by a Hill equation with U0 ¼ 123 ± pmolặmg)1, IC50 ẳ 82 pmolặmg)1, and a Hill coefficient of 1.25 ± 0.02 (v2 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 (U0 ẳ 135 pmolặmg)1, IC50 ẳ 47 pmolặmg)1, v2 ẳ 395) 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, M5I-1, specifically detected an intact band at 190 kDa which shifted to 160 kDa after treatment with peptide N-glycosidase F (PNGaseF) (Fig 5A) suggesting that MRP5 is N-glycosylated A protein with the same apparent molecular mass was also detected in doxorubicinresistant MOR/R and COR-L23/R lung tumour cells but at greatly reduced level in the doxorubicin-sensitive COR-L23/ P lung tumour cells (Fig 5B) The anti-MRP4 Ig detected an intact band at 170–180 kDa in human erythrocytes and MOR/R cells but not in COR-L23/R cells (Fig 5B) Discussion It is now well recognized that inside-out membrane vesicles prepared from human erythrocytes can take up both Ó FEBS 2003 cGMP transport in human erythrocytes (Eur J Biochem 270) 3703 Fig Inhibition of uptake of 3.3 lM cGMP by (A) MK-571, (B) cAMP, (C) glibenclamide and (D) clotrimazole (A) Inhibition by MK-571 The curve is the best fit assuming simple competition, U0 ¼ 130 pmolặmg)1, IC50 ẳ 0.38 0.01 lM, v2 ¼ 95 The Hill equation provides a closer fit with, U0 ẳ 120 pmolặmg)1, IC50 ẳ 0.48 0.02 lM, ni ¼ 1.10 ± 0.02 and v2 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 pmolỈmg)1) to the noninhibitable component (B) Inhibition by cAMP The curve shows the best fit for simple competition with U0 ẳ 108 pmolặmg)1, IC50 ¼ 296 ± 26 lM, v2 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 U0 ¼ 80 ± pmolỈmg)1, IC50 ¼ 2.8 ± 0.1 lM, v2 ¼ 250 Fits of the Hill equation and of simple competition plus a noninhibitable component of uptake did not produce significant improvements in the fit (F and P-values 3.26 & 0.12 and 5.0 & 0.07, respectively) The best fit for the noninhibitable component was 3.4 ± 0.3 pmolỈmg)1 (D) Inhibition by clotrimazole The curve is the best fit for simple competition U0 ẳ 97 pmolặmg)1, IC50 ¼ 24 ± lM, v2 ¼ 86 Fits of the Hill equation and of simple competition plus a noninhibitable component did not produce significant improvements in the fit (F and P-values of 4.4 & 0.1 and 4.7 & 0.1, respectively The best fit value of the Hill coefficient was less than (0.72) glutathione-conjugates, such as DNP-SG, and cyclic nucleotides, e.g cGMP, by rapid ATP-dependent transport processes In the present study, uptake of cGMP is shown to consist primarily of a low-affinity component with a maximum uptake (Umax) of 300–400 pmolỈmg)1Ỉmin)1 and a dissociation constant (Kd) in the range of 50–82 lM and possibly also a second high affinity component of uptake contributing less than 20% of the total trasnport even at low concentrations However, MK-571, glibenclamide, DNPSG, clotrimazole and cAMP all inhibit this uptake as if there were only a single component of transport (Figs 3C and 4) If present, the high-affinity component may correspond to the high-affinity transport previously reported In those studies [2,5] ATP-dependent cGMP uptake into inside-out membrane vesicles from human erythrocytes was found to have two components, a high affinity uptake with Umax1 ¼ 0.20.4 pmolặmg)1ặmin)1 and Kd1 ẳ 2.4 4.7 lM and a low afnity uptake with Umax2 ẳ 1.6 pmolặmg)1ặmin)1 and Kd2 ¼ 170 lM The maximum uptake rates in these studies were very low, being just over threefold higher than the background [2] The maximum uptake rate in the current study is two orders of magnitude higher The reasons for this remarkable difference are unclear though there may be several factors involved These include different osmolalities of the solutions used to measure uptake, differences in the methods of vesicle preparation, and possibly even differences in the profile of transporters present on the red cell membranes from different donors In the present study, the vesicles were resealed and assayed in low osmolality solution This contrasts with the previous study where the vesicles were resealed at low Ó FEBS 2003 3704 A Klokouzas et al (Eur J Biochem 270) Table Effect of MRP substrates, inhibitors and modulators on the ATP-dependent uptake of [3H]cGMP by inside-out human erythrocyte membrane vesicles Data represent mean ± SEM of n experiments The control uptake of [3H]cGMP (addition of dimethylsulfoxide only) for indomethacin, methotrexate and E217bG was 77.3 ± 5.2 pmolỈmg)1 The control uptake for the remaining drugs was 136.5 ± pmolỈ mg)1 Compound GSH Vincristine + mM GSH Calcein + mM GSH Indomethacin Methotrexate 17bE2G Taurocholic acid Concentration (lM) [3H]cGMP uptake (% control) n 500 1000 2000 4000 100 200 100 100 300 100 20 50 275 375 65 200 350 101.2 108.3 100.1 98.0 106.3 100.6 116.4 73.9 38.5 74.4 95.9 7.9 25.2 3.5 12.6 80.1 43.1 3 3 3 3 3 3 3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.8 7.1 1.9 1.2 7.6 3.2 1.1 1.8 3.4 1.8 3.7 5.6 2.8 1.1 3.7 2.1 1.1 osmolality but assayed in NaCl/Pi with an osmolality 10–100-fold higher To check these solutions as possible influencing factors, cGMP uptake in the current study was also measured using solutions as employed in the previous studies Under these conditions, uptake rates were lower, but only by a factor of two, i.e too small an effect to explain alone the discrepancies in results between the two studies Another possible explanation for the discrepancy is the presence of traces of calcium in the previous studies It was shown there that calcium can inhibit cGMP transport with an IC50 of about 40 lM and the inclusion of 100 lM 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 lM 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 vesiculation or stimulatory elements removed in the lengthy Steck and Kant procedure remains to be determined Certainly it has been proposed that the spontaneous vesiculation process may remove restrictive links with cytoskeletal elements [28] allowing more lateral mobility and thus the possibility of alterations in associations between membrane Table Effect of ion channel inhibitors on the ATP-dependent uptake of [3H]cGMP by inside-out human erythrocyte membrane vesicles Data represent mean ± SEM of n experiments The control level of [3H]cGMP uptake with dimethylsulfoxide (n ¼ 16) for furosemide, SITS, probenecid, staurosporine and Ro 31–8220 was in the range of 77–82 pmolỈmg)1 protein The control level of [3H]cGMP uptake with ethanol (n ¼ 18) for phloridzin, niflumic acid, and verapamil was in the range of 90–105 pmolỈmg)1 protein The control level of [3H]cGMP uptake for the remaining drugs was in the range of 129– 135 pmol mg)1 protein Compound Anion channel inhibitors Frusemide Niflumic acid Phloridzin SITS Probenecid Cation channel inhibitors BaCl2 CsCl 4-Aminopyridine Tetraethylammonium Cl Verapamil Verapamil + mM GSH PKC inhibitors Staurosporine Ro31–8220 Various Colchicine Imidazole Dideoxyforskolin Forskolin IBMX Concentration (lM) 0.5 10 20 50 [3H]cGMP uptake (%control) n 95.3 90.7 37.1 19.4 6.6 6.9 ± ± ± ± ± ± 2.3 3.1 2.1 2.6 0.9 1.3 6 10 20 50 105.2 45.6 21.4 5.4 5.6 ± ± ± ± ± 2.1 3.7 1.5 0.5 1.1 6 10 50 200 200 400 109.8 99.9 75.2 28.3 3.1 11.3 8.1 ± ± ± ± ± ± ± 3.3 4.7 4.2 2.3 0.9 0.7 1.1 5 10 3 400 1000 400 1000 500 10000 50 50 64.7 47.9 100.1 102.4 87.7 87.3 66.8 71.3 ± ± ± ± ± ± ± ± 3.2 1.6 2.7 3.1 4.6 3.4 1.7 3.5 3 3 3 3 2.9 ± 0.7 74.2 ± 2.3 3 98.4 94.1 95.3 66.7 16.5 3 10 10 50 100 100 100 100 ± ± ± ± ± 3.0 0.8 2.2 1.6 1.3 proteins The additional possibility that different donors possess different profiles of transporters on their red cell membranes is currently being explored In contrast to uptake of cGMP, ATP-dependent uptake of DNP-SG into inside-out erythrocyte membrane vesicles in the current study clearly possesses more than one component This has been noted also by Akerboom et al [7] and Pulaski et al [10] The observations are consistent with the presence of two components, one, a low-capacity Ó FEBS 2003 cGMP transport in human erythrocytes (Eur J Biochem 270) 3705 Fig 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) M5I-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 M5I-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 high-affinity component (Kd 1–10 lM) that predominates at concentrations below lM and another low-affinity component (> 100 lM) that is dominant at high concentrations The high-affinity component of DNP-SG transport, identified previously as being mediated by MRP1 [10–12], appears not to be involved in transport of cGMP (see below) However, the low affinity component of DNP-SG transport is inhibited by cGMP and may well be the transporter that mediates low-affininty cGMP uptake DNP-SG can indeed inhibit cGMP transport Furthermore, the apparent dissociation constant for DNP-SG uptake, kd2, and the IC50 for DNP-SG-mediated inhibition of cGMP uptake are approximately equal, i.e 65 lM compared with 82 lM Though the two values for cGMP uptake and for cGMP inhibition of DNP-SG uptake differ, i.e 50– 82 lM compared with 133 lM, it remains possible that a single transporter accounts for both uptakes as the interaction 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 As a wide variety of MRP transport inhibitors block cGMP and DNP-SG transport (Table and Fig 4), members of the MRP family are very plausible candidates for the transporters mediating ATP-dependent uptake of cGMP and DNP-SG into inside-out erythrocyte vesicles MRP1 and MRP5 have previously been detected on the membranes of human erythrocytes [8,9] MRP1 is known to mediate the high-affinity transport of DNP-SG [10–12] and MRP4 and MRP5 are known to transport cGMP [9,13,17] In the present study in addition to confirming the presence of MRP1 and MRP5 in erythrocyte membranes, we have detected the presence of MRP4 (see Fig 5) Although no cross-reactivity tests have been reported for anti-MRP4, the present finding that the anti-MRP4 Ig does not detect an immunoreactive band in COR-L23/R cells which express MRP1 and MRP5 suggests that this Ig does not cross-react with MRP1 or MRP5 Several lines of evidence show that MRP1 is most unlikely to be the transporter responsible for the transport of cGMP It has already been shown that cGMP does not inhibit transport of LTC4, an established high-affinity MRP1 substrate [4]; cGMP does not inhibit MRP1mediated transport of DNP-SG; and, verapamil inhibits cGMP uptake without any requirement for GSH (this study) while GSH is needed for its inhibition of MRP1mediated transport [12,51] Most convincingly, uptake of 3.3 lM cGMP is unaffected by the presence of the conformation-dependent monoclonal Ig against MRP1, QCRL-3, at 10 lgỈmL)1 which has been shown to block MRP1 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 relatively high-affinity trasnport with Km values in the range of 2–10 lM [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 [3H]cGMP uptake into inside-out vesicles MK-571, a leukotriene receptor antagonist, inhibited the cGMP transport with an IC50 value of 0.38 ± 0.01 lM 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 lM on cGMP transport attributed to MRP5 [9] Methotrexate and E217bG were found to inhibit the cGMP uptake in human erythrocyte vesicles with E217bG at 65 lM inhibiting about 90% and methotrexate inhibiting about 75% at 275 lM and completely at 375 lM These compounds are established MRP4 substrates with Km values around 220 lM for methotrexate [19,39], and 30 lM for E217bG [39,3,4] At present, methotrexate and E217bG appear to be substrates of MRP4 [13,39] and not of MRP5 cAMP, which has been shown to be transported by both MRP4 and MRP5 [9,13], inhibited all of the cGMP uptake with an estimated IC50 value of 315 ± 70 lM The ratio of this value to the apparent Kd value for cGMP, about 5, is Ó FEBS 2003 3706 A Klokouzas et al (Eur J Biochem 270) consistent with the value, 5, reported for MRP4 [13] but not the value, 380, reported for MRP5 [9] Taurocholic acid, an established substrate for MRP1, MRP2, and MRP3, had only a weak inhibitory effect on the cGMP uptake (about 20% at 200 lM and about 60% at 350 lM) Interpretation of such inhibition is difficult given that taurocholic acid would probably be acting as a detergent at high concentrations Glibenclamide was found to be a very potent inhibitor of cGMP transport in the human erythrocyte membrane vesicles with an estimated IC50 value of 1.9 ± 0.1 lM This was the second most potent of the inhibitors tested against the cGMP transporter Glibenclamide has been shown to interact with several ABC proteins including the sulfonylurea receptor [42,52], CFTR [40], and more recently, P-glycoprotein and MRP1 [41,53], blocking their function Given that glibenclamide inhibits many ABC transporters, its action serves merely to point to an ABC transporter as being responsible for cGMP transport but not to identify which ABC transporter it is Several other compounds including forskolin, an activator of adenylate cyclase, and IBMX, a nonspecific phosphodiesterase inhibitor with structural similarity to cGMP, also inhibited the cGMP uptake Forskolin at a tested concentration of 100 lM inhibited the cGMP transport by about 35% while its inactive analogue, 1,9-dideoxyforskolin, 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 structurally related compound, IBMX, currently used as a nonspecific phosphodiesterase inhibitor, proved an effective inhibitor of cGMP transport at 100 lM The PKC inhibitors, staurosporine and Ro 31–8220, inhibited the cGMP uptake Staurosporine completely inhibited the cGMP uptake at 10 lM while at the same concentration Ro 31– 8220 inhibited by about 25% The greater potency of staurosporine is consistent with a recently proposed mechanism for its action: interaction with the ATP binding sites of the transporter inhibiting energy-dependent drug efflux activity [49] The major characteristics of the cGMP transporter in human erythrocytes, as described in the present study, are inhibition by MK-571, glibenclamide, E217bG, methotrexate, DNP-SG and cAMP This inhibitor profile matches well with that for MRP4 At present, five distinguishing features exist between human MRP4 and MRP5 First, MRP4 is inhibited by MK-571 [39] while MRP5 is not [9] Second, MRP4 transports methotrexate [39] but MRP5 does not confer resistance to this drug suggesting no transport [55] Third, human MRP4 transports 17bE2G [13] but human MRP5 appears not to so [9] Fourthly, cAMP and cGMP interact with MRP4 at similar concentrations [13] while the apparent KD for cAMP with MRP5 is much higher than that for cGMP [9] In summary, using inside-out membrane vesicles prepared from human erythrocytes by a spontaneous, one-step vesiculation process, a dominant low affinity component of cGMP transport with Kd value in the region of 50–80 lM has been identified This transport is completely inhibitable by MK-571, glibenclamide, clotrimazole, cAMP and DNP-SG, consistent with the idea of a single transporter being responsible It is also markedly inhibited by methotrexate All of these and cGMP block low affinity [3H]DNPSG 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 characteristics 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 M5I-1 Ig and Dr M Turner for the MK-571 References Flo, K., Hansen, M., Orbo, A., Kjorstad, K.E., Maltau, J.M & Sager, G (1995) Effect of probenecid, verapamil and progesterone on the concentration-dependent and temperature-sensitive human erythrocyte uptake and export of guanosine 3¢,5¢-cyclic monophosphate (cGMP) Scand J Clin Lab Inv 55, 715–721 Schultz, C., Vaskinn, S., Kildalsen, H & Sager, G (1998) Cyclic AMP stimulates the cyclic GMP egression pump in human erythrocytes: Effects of probenecid, verapamil, progesterone, theophylline, IBMX, forskolin, and cyclic AMP on cyclic GMP uptake and association to inside-out vesicles Biochemistry 37, 1161–1166 Vaskinn, S., Sundkvist, E., Jaeger, R & Sager, G (1999) The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of H-3-cGMP to inside-out vesicles from human erythrocytes Mol Membr Biol 16, 181–188 Sundkvist, E., Jaeger, R & Sager, G (2000) Leukotriene C4 (LTC4) does not share a cellular efflux mechanism with cGMP: Characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes Biochim Biophys Acta: Biomembranes 1463, 121–130 Sager, G., Orbo, A., Pettersen, R.H & Kjorstad, K.E (1996) Export of guanosine 3¢,5¢-cyclic monophosphate (cGMP) from human erythrocytes characterized by inside-out membrane vesicles Scand J Clin Lab Inv 56, 289–293 Eckert, K.G & Eyer, P (1986) Formation and transport of xenobiotic glutathione-S-conjugates in red cells Biochem Pharmacol 35, 325–329 Akerboom, T.P.M., Bartosz, G & Sies, H (1992) Low- and highK (m) transport of dinitrophenyl glutathione in inside out vesicles from human erythrocytes Biochim Biophys Acta: Biomembranes 1103, 115–119 Flens, M.J., Zaman, G.J., Van de Valk, P., Izquierdo, M.A., Schroeijers, A.B., Scheffer, G.L., Van der Groep, P., de Haas, M., Meijer, C.J & Scheper, R.J (1996) Tissue distribution of the multidrug resistance protein Am J Pathol 148, 1237–1247 Jedlitschky, G., Burchell, B & Keppler, D (2000) The multidrug resistance protein functions as an ATP- dependent export pump for cyclic nucleotides J Biol Chem 275, 30069–30074 10 Pulaski, L., Jedlitschky, G., Leier, I., Buchholz, U & Keppler, D (1996) Identification of the multidrug-resistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes Eur J Biochem 241, 644–648 11 Wijnholds, J., Evers, R., Van Leusden, M.R., Mol, C.A., Zaman, G.J., Mayer, U., Beijnen, J.H., Van der Valk, M., Krimpenfort, P Ó FEBS 2003 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 cGMP transport in human erythrocytes (Eur J Biochem 270) 3707 & Borst, P (1997) Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein Nat Med 3, 1275–1279 Klokouzas, A., Barrand, M.A & Hladky, S.B (2001) Effects of clotrimazole on transport mediated by multidrug resistance associated protein (MRP1) in human erythrocytes and tumour cells Eur J Biochem 268, 6569–6577 Chen, Z.S., Lee, K & Kruh, G.D (2001) Transport of cyclic nucleotides and estradiol 17-b-D-glueuronide by multidrug resistance protein – Resistance to 6-mercaptopurine and 6-thioguanine J Biol Chem 276, 33747–33754 Lai, L.Q & Tan, T.M (2002) Role of glutathione in the multidrug resistance protein (MRP4/ABCC4) mediated efflux of cAMP and resistance to purine analogues Biochem J 361, 497–503 Boadu, E., Vaskinn, S., Sundkvist, E., Jaeger, R & Sager, G (2001) Inhibition by guanosine cyclic monophosphate (cGMP) analogues of uptake of H-3 3¢,5¢-cGMP without stimulation of ATPase activity in human erythrocyte inside-out vesicles Biochem Pharmacol 62, 425–429 Sundkvist, E., Jaeger, R & Sager, G (2002) Pharmacological characterization of the ATP-dependent low K-m guanosine 3¢,5¢cyclic monophosphate (cGMP) transporter in human erythrocytes Biochem Pharmacol 63, 945–949 Wielinga, P.R., Van Der Heijden, I., Reid, G., Beijnen, J.H., Wijnholds, J & Borst, P (2003) Characterization of the MRP4and MRP5-mediated transport of cyclic nucleotides from intact cells J Biol Chem Scheffer, G.L., Kool, M., Heijn, M., de Haas, M., Pijnenborg, A., Wijnholds, J., van Helvoort, A., de Jong, M.C., Hooijberg, J.H., Mol, C., van der Linden, M., de Vree, J.M.L., van der Valk, P., Elferink, R., Borst, P & Scheper, R.J (2000) Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies Cancer Res 60, 5269–5277 Lee, K., Klein-Szanto, A.J.P & Kruh, G.D (2000) Analysis of the MRP4 drug resistance profile in transfected NIH3T3 cells J Natl Cancer Inst 92, 1934–1940 Ishikawa, T (1989) ATP/Mg2+-dependent cardiac transport system for glutathione S-conjugates A study using rat heart sarcolemma vesicles J Biol Chem 264, 17343–17348 Awasthi, Y.C., Garg, H.S., Dao, D.D., Partridge, C.A & Srivastava, S.K (1981) Enzymatic conjugation of erythrocyte glutathione with 1-chloro-2,4-dintrobenzene: the fate of glutathione conjugate in ertythrocytes and the effect of glutathione depletion on hemoglobin Blood 58, 733–738 Twentyman, P.R., Fox, N.E., Wright, K.A & Bleehen, N.M (1986) Derivation and preliminary characterization of adriamycin resistant lines of human lung cancer cells Br J Cancer 53, 529–537 Barrand, M.A., Heppell-Parton, A.C., Wright, K.A., Rabbitts, P.H & Twentyman, P.R (1994) A 190k protein overexpressed in non-P-glycoprotein containing MDR cells and its relation to the MRP gene J Natl Cancer Inst 86, 110–117 Kool, M., De Haas, M., Scheffer, G.L., Scheper, R.J., Van Eijk, M.J.T., Juijn, J.A., Baas, F & Borst, P (1997) Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines Cancer Res 57, 3537–3547 Ishikawa, T., Wright, C.D & Ishizuka, H (1994) GS-X pump is functionally overexpressed in cis-diamminedichloroplatinum (ii) resistant human leukemia HL-60 cells and down-regulated by celldifferentiation J Biol Chem 269, 29085–29093 Lew, V.L., Muallem, S & Seymour, C.A (1982) Properties of the Ca2+-activated K+ channel in one-step inside-out vesicles from human red cell membranes Nature 296, 742–744 27 Lew, V.L & Seymour, C.A (1982) Cation transport in one-step inside-out vesicles from red cell membranes In Techniques in the Life Sciences, Biochemistry Vol B4/1 (Hesketh, T.R., Korenburg, H.L., Metcalfe, J.C., Northcote, D.H., Pogson, C.I and Tipton, K.F., eds.), pp 1–13 Elsevier, Holland 28 Lew, V.L., Hockaday, A., Freeman, C.J & Bookchin, R.M (1988) Mechanism of spontaneous inside-out vesiculation of red cell membranes J Cell Biol 106, 1893–1901 29 Ellman, G.L., Courtney, K.D., Andres, V.J & Featherstone, R.M (1961) A new and rapid colorimetric determination of acetylcholinesterase activity Biochem Pharmacol 7, 88–95 30 Steck, T.L & Kant, J.A (1974) Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes Methods Enzymol 31, 172–180 31 Schrier, S.L., Ben-Bassat, I., Junga, I., Seeger, M & Grumet, F.C (1975) Characterization of erythrocyte membrane-associated enzymes (glyceraldehyde-3-phosphate dehydrogenase and phosphoglyceric kinase) J Lab Clin Med 85, 797–810 32 Pratt, W.B & Taylor, P (1990) Principles of Drug Action The Basis of Pharmacology, 3rd edn Churchill Livingstone, New York 33 Rodbard, D (1974) Statistical quality control and routine data processing for radioimmunoassays and immunoradiometric assays Clin Chem 20, 1255–1270 34 Maley, F., Trimble, R.B., Tarentino, A.L & Plummer, T.H (1989) Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases Anal Biochem 180, 195–204 35 Hipfner, D.R., Mao, Q., Qiu, W., Leslie, E.M., Gao, M., Deeley, R.G & Cole, S.P.C (1999) Monoclonal antibodies that inhibit the transport function of the 190-kDa multidrug resistance protein, MRP Localization of their epitopes to the nucleotide-binding domains of the protein J Biol Chem 274, 15420–15426 36 Gekeler, V., Ise, W., Sanders, K.H., Ulrich, W.R & Beck, J (1995) The leukotriene LTD (4) receptor antagonist MK571 specifically modulates MRP associated multidrug-resistance Biochem Biophys Res Comm 208, 345–352 37 Jedlitschky, G., Leier, I., Buchholz, U., Center, M & Keppler, D (1994) ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein Cancer Res 54, 4833–4836 38 Chen, Z.S., Kawabe, T., Ono, M., Aoki, S., Sumizawa, T., Furukawa, T., Uchiumi, T., Wada, M., Kuwano, M & Akiyama, S (1999) Effect of multidrug resistance-reversing agents on transporting activity of human canalicular multispecific organic anion transporter Mol Pharmacol 56, 1219–1228 39 Chen, Z.S., Lee, K., Walther, S., Raftogianis, R.B., Kuwano, M., Zeng, H & Kruh, G.D (2002) Analysis of methotrexate and folate transport by multidrug resistance protein (ABCC4): MRP4 is a component of the methotrexate efflux system Cancer Res 62, 3144–3150 40 Schultz, B.D., DeRoos, A.D.G., Venglarik, C.J., Singh, A.K., Frizzell, R.A & Bridges, R.J (1996) Glibenclamide blockade of CFTR chloride channels Am J Physiol 15, L192–L200 41 Golstein, P.E., Boom, A., van Geffel, J., Jacobs, P., Masereel, B & Beauwens, R (1999) P-glycoprotein inhibition by glibenclamide and related compounds Pflugers Archiv Eur J Physiol 437, 652–660 42 Aguilar-Bryan, L., Nichols, C.G., Wechsler, S.W., Clement, J.P., Boyd, A.E., Gonzalez, G., Herrerasosa, H., Nguy, K., Bryan, J & Nelson, D.A (1995) Cloning of the beta-cell high-affinity sulfonylurea receptor – a regulator of insulin secretion Science 268, 423–426 43 Gros, L., Virsolvy, A., Salazar, G., Bataille, D & Blache, P (1999) Characterization of low-affinity binding sites for glibenclamide on Ó FEBS 2003 3708 A Klokouzas et al (Eur J Biochem 270) 44 45 46 47 48 49 the Kir6.2 subunit of the beta-cell K-ATP channel Biochem Biophys Res Comm 257, 766–770 Hollo, Z., Homolya, L., Hegedus, T & Sarkadi, B (1996) Transport properties of the multidrug resistance-associated protein (MRP) in human tumour cells FEBS Lett 383, 99– 104 Loe, D.W., Almquist, K.C., Deeley, R.G & Cole, S.P.C (1996) Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles: Demonstration of glutathione-dependent vincristine transport J Biol Chem 271, 9675–9682 Loe, D.W., Deeley, R.G & Cole, S.P.C (1998) Characterization of vincristine transport by the M fi 190,000 multidrug resistance protein (MRP): Evidence for cotransport with reduced glutathione Cancer Res 58, 5130–5136 Bagrij, T & Barrand, M.A (2000) Characteristics of reduced glutathione efflux from human lung tumour cells contraining different amounts of multidrug resistance-associated protein, MRP1 Br J Pharmacol 129, 266P Beltman, J., McCormick, F & Cook, S.J (1996) The selective protein kinase C inhibitor, Ro-31–8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase J Biol Chem 271, 27018–27024 Conseil, G., Perez-Victoria, J.M., Jault, J.M., Gammaro, F., Goffeau, A., Hofmann, J & Di Pietro, A (2001) Protein kinase C 50 51 52 53 54 55 effectors bind to multidrug ABC transporters and inhibit their activity Biochemistry 40, 2564–2571 Budworth, J., Davies, R., Malkhandi, J., Gant, T.W., Ferry, D.R & Gescher, A (1996) Comparison of staurosporine and four analogues: Their effects on growth, rhodamine 123 retention and binding to P- glycoprotein in multidrug-resistant MCF-7/Adr cells Br J Cancer 73, 1063–1068 Loe, D.W., Deeley, R.G & Cole, S.P.C (2000) Verapamil stimulates glutathione transport by the 190-kDa multidrug resistance protein (MRP1) J Pharmacol Exp Ther 293, 530–538 Ashcroft, S.J.H (2000) The beta-cell K-ATP channel J Membr Biol 176, 187–206 Payen, L., Delugin, L., Courtois, A., Trinquart, Y., Guillouzo, A & Fardel, O (2001) The sulphonylurea glibenclamide inhibits multidrug resistance protein (MRP1) activity in human lung cancer cells Br J Pharmacol 132, 778–784 Morris, D.I., Speicher, L.A., Ruoho, A.E., Tew, K.D & Seamon, K.B (1991) Interaction of forskolin with the P-glycoprotein multidrug transporter Biochemistry 30, 8371–8379 Wijnholds, J., Mol, C., van Deemter, L., de Haas, M., Scheffer, G.L., Baas, F., Beijnen, J.H., Scheper, R.J., Hatse, S., De Clercq, E., Balzarini, J & Borst, P (2000) Multidrug-resistance protein is a multispecific organic anion transporter able to transport nucleotide analogs Proc Natl Acad Sci USA 97, 7476–7481  ... and that vincristine inhibits the high affinity DNP-SG transport in the presence but not in the absence of GSH [12,45,46] Thus the effect of vincristine on the cGMP uptake was also tested in the. .. these and cGMP block low affinity [3H]DNPSG 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. .. 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