Modulation of MRP1-like efflux activity in human erythrocytes caused by membrane perturbing agents

The effect of membrane perturbing agents on the efflux (37°C, 60 min) of the fluorescent probe 2′, 7′-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was studied. Several anionic amphiphiles (detergents) markedly inhibited BCPCF efflux (IC₅₀≤?40 μM). Most zwitter-ionic amphiphiles were inefficient inhibitors. Non-ionic and cationic amphiphiles had minor effects or increased efflux. Of the aliphatic inhibitors, C₁₂-homologues were the most efficient. Hexanol, ethanol, methyl-β-cyclodextrin (MβCD) and diamide (+ washing) did not influence BCPCF efflux. It is suggested that amphiphiles affect BCPCF efflux by modulating multi-drug resistance protein 1 (MRP1, ABCC1) activity. A negative charge of amphiphiles is essential for the inhibitory effect, while alkyl chain length modulates inhibition. MRP1-mediated BCPCF efflux appears to be relatively insensitive to non-specific plasma membrane modification.     

Flow cytometric monitoring of multidrug drug resistance protein 1 (MRP1/ABCC1) -mediated transport of 2',7'-bis-(3-carboxypropyl)-5-(and-6)- carboxyfluorescein (BCPCF) into human erythrocyte membrane inside-out vesicles

The presence of human multidrug resistance protein 1 (MRP1/ABCC1) in the human erythrocyte membrane is well established. In the present study, flow cytometric monitoring is introduced to identify MRP1 as the main transporter of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) in the erythrocyte membrane and to facilitate inhibition and kinetic studies of MRP1-mediated transport. The ATP-dependent transport of BCPCF into human erythrocyte inside-out vesicles and, for comparison, into MRP1-expressing Sf9 cell membrane inside-out vesicles were studied. The MRP1-specific monoclonal antibody, QCRL-3 and the MRP1 inhibitor, MK-571 strongly decreased the uptake of BCPCF into both erythrocyte and MRP1-expressing Sf9 cell membrane inside-out vesicles. The inhibition profiles of cyclosporin A, verapamil, benzbromarone, and probenecid in erythrocyte membrane vesicles were typical for MRP1-mediated transport. Furthermore, kinetic constants K(m) and V(max) of BCPCF transport into erythrocyte membrane inside-out vesicles were determined in the absence and in the presence of selected inhibitors (MK-571, cyclosporin A, benzbromarone and verapamil). The presented results identified MRP1 as the major transporter of BCPCF in the human erythrocyte membrane and showed for the first time that the active transport of fluorescent substrate into inside-out vesicles can be monitored by flow cytometry.     

Flow cytometric monitoring of multidrug drug resistance protein 1 (MRP1/ABCC1) -mediated transport of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)- carboxyfluorescein (BCPCF) into human erythrocyte membrane inside-out vesicles

The presence of human multidrug resistance protein 1 (MRP1/ABCC1) in the human erythrocyte membrane is well established. In the present study, flow cytometric monitoring is introduced to identify MRP1 as the main transporter of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) in the erythrocyte membrane and to facilitate inhibition and kinetic studies of MRP1-mediated transport. The ATP-dependent transport of BCPCF into human erythrocyte inside-out vesicles and, for comparison, into MRP1-expressing Sf9 cell membrane inside-out vesicles were studied. The MRP1-specific monoclonal antibody, QCRL-3 and the MRP1 inhibitor, MK-571 strongly decreased the uptake of BCPCF into both erythrocyte and MRP1-expressing Sf9 cell membrane inside-out vesicles. The inhibition profiles of cyclosporin A, verapamil, benzbromarone, and probenecid in erythrocyte membrane vesicles were typical for MRP1-mediated transport. Furthermore, kinetic constants K_m and V_max of BCPCF transport into erythrocyte membrane inside-out vesicles were determined in the absence and in the presence of selected inhibitors (MK-571, cyclosporin A, benzbromarone and verapamil). The presented results identified MRP1 as the major transporter of BCPCF in the human erythrocyte membrane and showed for the first time that the active transport of fluorescent substrate into inside-out vesicles can be monitored by flow cytometry.     

Modulation of MRP1 protein transport by plant, and synthetically modified flavonoids

The influence of novel synthetic and plant origin flavonoids on activity of multidrug resistance-associated protein (MRP1) was investigated in human erythrocytes used as a cell model expressing MRP1 in plasma membrane. The fluorescent probe, BCPCF (2', 7'-bis-(3-carboxy-propyl)-5-(and-6)-carboxyfluorescein), was applied as a substrate for MRP1 multidrug resistance transporter. The effect of compounds belonging to different classes of natural flavonoids: flavone, flavonol, isoflavones and flavanolignan was compared with action of new synthetic derivatives of genistein. Most of the flavonoids showed strong or moderate ability to inhibit transport carried out by MRP1. Inhibitory properties of flavonoids were compared to the effects of indomethacin, probenecid and MK-571 known as MRP1 inhibitors. Studying the influence of new synthetic genistein derivatives on BCPCF transport we have found that the presence of hydrophobic groups substituting hydrogen of hydroxyl group at the position 4' in ring B of isoflavone is more important for inhibitory properties than hydrophobic substitution at the position 7 in ring A. In case of naturally occurring isoflavones the replacement of hydrogen at position 4' by hydrophobic ring structure seems also to be favourable for inhibition potency.     

Phenothiazine maleates stimulate MRP1 transport activity in human erythrocytes

The expression of multidrug resistance-associated protein (MRP1) results in ATP-dependent reduction of drugs' concentration in cancer cells, i.e., multidrug resistance (MDR). Since the majority of projects are concentrated on the search of the new MDR modulators, there are very few reports on drug-induced stimulation of MDR transporters activity. In the present work, by means of functional fluorescence assay we have shown that MRP1-mediated efflux of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) out of human erythrocytes is stimulated by phenothiazine maleates that have been already identified as P-glycoprotein inhibitors. Phenothiazine maleates-induced stimulation of ATP-dependent uptake of 2',7'-bis-(3-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) into inside-out membrane vesicles prepared from erythrocyte membranes has been also demonstrated. Moreover, it was shown that phenothiazine maleates exerted stimulating effect on ATPase activity measured in erythrocyte membranes. To our best knowledge, this report is the first one demonstrating that compounds able to inhibit transport activity of P-glycoprotein can stimulate MRP1 transporter. We conclude that phenothiazine maleates probably exert their stimulatory effect on MRP1 by direct interaction with the protein at the site different from the substrate binding site.     

Permeability alterations and antihaemolysis induced by amphiphiles in human erythrocytes

In an attempt to define the parameters in amphiphilic molecules important for their interaction with the erythrocyte membrane, the effects of cationic, anionic, zwitterionic and nonionic amphiphilic agents (C10-C16) on osmotic fragility and transport of potassium and phosphate in human erythrocytes were studied. All the amphiphiles protected the erythrocytes against hypotonic haemolysis. Half-maximum protection occurred at a concentration which was about 15% of that inducing 50% haemolysis. The concentrations of amphiphiles required to induce protection or haemolysis were related to the length of the alkyl chain in a way indicating that a membrane/aqueous phase partition is the mechanism whereby the amphiphile monomers intercalate into the membrane. At antihaemolytic concentrations all the amphiphiles increased potassium efflux and passive potassium influx. The increase in the fluxes was about the same in both directions through the membrane and there were no clear differences in the effects of the different amphiphilic derivatives at equi-protecting concentrations. Active potassium influx was decreased by cationic, zwitterionic and non-ionic amphiphiles. The ability of the amphiphiles to inhibit the influx was not related to the length of the alkyl chain. Anionic amphiphiles had no or only a weak stimulatory effect on the influx. Phosphate efflux was reduced by all the amphiphiles. The inhibitory potency of the different amphiphiles decreased in the following order; anionic greater than zwitterionic, non-ionic greater than cationic. Short-chained amphiphiles were more potent inhibitors than long-chained. The possible participation of non-bilayer phases (mixed inverted micelles) in the intercalation of amphiphiles into the membrane is discussed.     

Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.     

Evidence for a multidrug resistance-associated protein 1 (MRP1)-related transport system in cultured rat liver biliary epithelial cells

Cellular accumulation and efflux of the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CF) were studied in rat liver SDVI cells thought to derive from primitive bile ductules, in order to characterize carrier-related membrane transport of organic anions in epithelial cells. Probenecid, a common blocker of anion transport, was found to strongly enhance CF levels in SDVI cells in a dose-dependent manner through inhibition of dye efflux. Such an outwardly-directed transport was demonstrated to be temperature-dependent and down-regulated by various metabolic inhibitors, therefore outlining its requirement for energy; it was shown to be Na+- and membrane potential-independent and inhibited by anionic drugs such as indomethacin, indoprofen and rifamycin B. These functional features are closed to those described for multidrug resistance-associated protein 1 (MRP1) that was furthermore demonstrated, in contrast to P-glycoprotein, to be expressed in SDVI cells and to lower CF accumulation in MRP1-overexpressing drug-resistant tumor cells. These data therefore suggest that active membrane transport of organic anions such as CF occurs in epithelial cells like cultured liver biliary SDVI cells through a MRP1-related efflux system.     

Function of MRP1/ABCC1 is not dependent on cholesterol or cholesterol-stabilized lipid rafts

MRP1 (multidrug-resistance-related protein 1)/ABCC1 (ATP-binding cassette transporter C1) has been localized in cholesterol-enriched lipid rafts, which suggests a role for these lipid rafts and/or cholesterol in MRP1 function. In the present study, we have shown for the first time that nearly complete oxidation of free cholesterol in the plasma membrane of BHK-MRP1 (MRP1-expressing baby hamster kidney) cells did not affect MRP1 localization in lipid rafts or its efflux function, using 5-carboxyfluorescein diacetate as a substrate. Inhibition of cholesterol biosynthesis, using lovastatin in combination with RO 48-8071, an inhibitor of oxidosqualene cyclase, resulted in a shift of MRP1 out of lipid raft fractions, but did not affect MRP1-mediated efflux in Neuro-2a (neuroblastoma) cells. Short-term methyl-β-cyclodextrin treatment was equally effective in removing free cholesterol from Neuro-2a and BHK-MRP1 cells, but affected MRP1 function only in the latter. The kinetics of loss of both MRP1 efflux function and lipid raft association during long-term methyl-β-cyclodextrin treatment did not match the kinetics of free cholesterol removal in both cell lines. Moreover, MRP1 activity was measured in vesicles consisting of membranes isolated from BHK-MRP1 cells using the substrate cysteinyl leukotriene C4 and was not changed when the free cholesterol level of these membranes was either decreased or increased. In conclusion, MRP1 activity is not correlated with the level of free cholesterol or with localization in cholesterol-dependent lipid rafts.     

Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cells.

Clotrimazole has been shown to have potent anti-malarial activity in vitro, one possible mechanism being inhibition of oxidized glutathione (GSSG) export from the infected human red blood cells or from the parasite itself. Efflux of GSSG from normal erythrocytes is mediated by a high affinity glutathione S-conjugate transporter. This paper shows that transport of the model substrate, 3 microm dinitrophenyl S-glutathione, across erythrocyte membranes is inhibited by multidrug resistance-associated protein 1 (MRP1)-specific antibody, QCRL-3, strongly suggesting that the high affinity transport is mediated by MRP1. The rates of transport observed with membrane vesicles prepared from erythrocytes or from multidrug resistant tumour cells show a similar pattern of responses to applied reduced glutathione, GSSG and MRP1 inhibitors (indomethacin, MK571) further supporting the conclusion that the high affinity transporter is MRP1. In both erythrocytes and MRP1-expressing tumour cells, MRP1-associated transport is inhibited by clotrimazole over the range 2-20 microm, and the inhibitory effect leads to increases in accumulation of MRP1 substrates, vincristine and calcein, and decreases in calcein efflux from intact MRP1-expressing human tumour cells. It also results in increased sensitivity to daunorubicin of the multidrug resistant cells, L23/R but not the sensitive parent L23/P cells. These results demonstrate that clotrimazole can inhibit the MRP1 which is present in human erythrocytes, an effect that may contribute to, though not fully account for, its anti-malarial action.     

Mechanism of RPE cell death in α-crystallin deficient mice: a novel and critical role for MRP1-mediated GSH efflux

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.     

Double-edged sword of chemosensitizer: increase of multidrug resistance protein (MRP) in leukemic cells by an MRP inhibitor probenecid

The multidrug resistance protein (MRP) is a drug efflux membrane pump conferring multidrug resistance to tumor cells. Clinical trials have been undertaken to improve the effectiveness of chemotherapy by adding an MRP inhibitor to the treatment regimen. This study attempted not only to determine novel resistance mechanisms in MRP-overexpressing AML cells (AML-2/DX100) by chronic exposure to doxorubicin in the presence of an MRP inhibitor probenecid but also to find out whether probenecid could increase MRP levels. AML-2/DXPBA cultured in the presence of probenecid (600 microM) and doxorubicin (100 ng/ml) showed a higher level of the multidrug resistance (MDR) phenotype when compared to AML-2/DX100. AML-2/DXPBA showed increased levels of MRP compared to those of AML-2/DX100. Probenecid increased the MRP levels without an increase in MRP mRNA in AML-2/WT in both a time- and dose-dependent manner. Of the MRP inhibitors including probenecid, ofloxacin, erythromycin, and rifampicin used in this study, only probenecid showed a marked chemosensitizing effect in AML-2/DX100 but not in HL-60/Adr, suggesting that the chemosensitizing effects of the MRP inhibitors vary according to the type of resistant cells. The maximum noncytotoxic concentrations of these MRP inhibitors increased the MRP levels to various degrees in both AML-2/WT and HL-60/WT. However, the chemosensitizing effects of the MRP inhibitors were not correlated with their MRP-increasing effects. Altogether, MRP inhibitors such as probenecid have been shown to function as a double-edged sword, indicating that they are not only an effective chemosensitizer of MRP-associated MDR tumor cells but also an MRP activator. Therefore caution should be taken whenever using MRP inhibitors to reverse MRP-mediated multidrug resistance in clinical cancer chemotherapy as well as when used to inhibit MRP expression in vitro.     

Effects of nonionic amphiphiles at sublytic concentrations on the erythrocyte membrane

The interactions of octaethyleneglycol alkylethers (C10-C16), pentaethyleneglycol dodecylether, and dodecyl D-maltoside with the human erythrocyte membrane were studied. All the amphiphiles protected erythrocytes against hypotonic haemolysis. At concentrations where the amphiphiles protected erythrocytes against hypotonic haemolysis they reduced phosphate efflux. The potency of the amphiphiles, at equiprotecting concentrations, was correlated negatively to the length of the alkyl chain. Potassium fluxes were increased by all the amphiphiles at protective concentrations. The relative potency of the amphiphiles varied but it was not simply related to the length of the alkyl chain. The only amphiphile affecting active potassium influx was octaethyleneglycol decylether which induced a slight decrease. It is concluded that the increase in passive cation fluxes caused by the amphiphiles is due to an increased permeability of the lipid bilayer induced through a nonspecific interaction of the amphiphiles with the bilayer. The effect of the amphiphiles on ion transport mediated by membrane proteins is proposed to be due to an alteration of the state of the transporting protein.     

Transport of antimony salts by Arabidopsis thaliana protoplasts over-expressing the human multidrug resistance-associated protein 1 (MRP1/ABCC1)

ABC transporters from the multidrug resistance-associated protein (MRP) subfamily are glutathione S-conjugate pumps exhibiting a broad substrate specificity illustrated by numerous xenobiotics, such as anticancer drugs, herbicides, pesticides and heavy metals. The engineering of MRP transporters into plants might be interesting either to reduce the quantity of xenobiotics taken up by the plant in the context of “safe-food” strategies or, conversely, in the development of phytoremediation strategies in which xenobiotics are sequestered in the vacuolar compartment. In this report, we obtained Arabidopsis transgenic plants overexpressing human MRP1. In these plants, expression of MRP1 did not increase plant resistance to antimony salts (Sb(III)), a classical glutathione-conjugate substrate of MRP1. However, the transporter was fully translated in roots and shoots, and targeted to the plasma membrane. In order to investigate the functionality of MRP1 in Arabidopsis, mesophyll cell protoplasts (MCPs) were isolated from transgenic plants and transport activities were measured by using calcein or Sb(III) as substrates. Expression of MRP1 at the plasma membrane was correlated with an increase in the MCPs resistance to Sb(III) and a limitation of the metalloid content in the protoplasts due to an improvement in Sb(III) efflux. Moreover, Sb(III) transport was sensitive to classical inhibitors of the human MRP1, such as MK571 or glibenclamide. These results demonstrate that a human ABC transporter can be functionally introduced in Arabidopsis, which might be useful, with the help of stronger promoters, to reduce the accumulation of xenobiotics in plants, such as heavy metals from multi-contaminated soils.     

Loss of multidrug resistance protein 1 expression and folate efflux activity results in a highly concentrative folate transport in human leukemia cells

We studied the molecular basis of the up to 46-fold increased accumulation of folates and methotrexate (MTX) in human leukemia CEM-7A cells established by gradual deprivation of leucovorin (LCV). CEM-7A cells consequently exhibited 10- and 68-fold decreased LCV and folic acid growth requirements and 23-25-fold hypersensitivity to MTX and edatrexate. Although CEM-7A cells displayed a 74-86-fold increase in the reduced folate carrier (RFC)-mediated influx of LCV and MTX, RFC overexpression per se cannot induce a prominently increased folate/MTX accumulation because RFC functions as a nonconcentrative anion exchanger. We therefore explored the possibility that folate efflux activity mediated by members of the multidrug resistance protein (MRP) family was impaired in CEM-7A cells. Parental CEM cells expressed substantial levels of MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and breast cancer resistance protein were undetectable. In contrast, CEM-7A cells lost 95% of MRP1 levels while retaining parental expression of MRP4 and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the [(3)H]folic acid efflux rate constant, which was identical to that obtained with parental CEM cells, when their folic acid efflux was blocked (78%) with probenecid. Furthermore, when compared with parental CEM, CEM-7A cells accumulated 2-fold more calcein fluorescence. Treatment of parental cells with the MRP1 efflux inhibitors MK571 and probenecid resulted in a 60-100% increase in calcein fluorescence. In contrast, these inhibitors failed to alter the calcein fluorescence in CEM-7A cells, which markedly lost MRP1 expression. Replenishment of LCV in the growth medium of CEM-7A cells resulted in resumption of normal MRP1 expression. These results establish for the first time that MRP1 is the primary folate efflux route in CEM leukemia cells and that the loss of folate efflux activity is an efficient means of markedly augmenting cellular folate pools. These findings suggest a functional role for MRP1 in the maintenance of cellular folate homeostasis.     

Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes

Recent studies indicate that members of the multidrug-resistance protein (MRP) family belonging to ATP binding cassette type C (ABCC) membrane proteins extrude cyclic nucleotides from various cell types. This study aimed to determine whether MRP proteins regulate cardiac cAMP homeostasis. Here, we demonstrate that MRP4 is the predominant isoform present at the plasma membrane of cardiacmyocytes and that it mediates the efflux of cAMP in these cells. MRP4-deficient mice displayed enhanced cardiac myocyte cAMP formation, contractility, and cardiac hypertrophy at 9 mo of age, an effect that was compensated transiently by increased phosphodiesterase expression at young age. These findings suggest that cAMP extrusion via MRP4 acts together with phosphodiesterases to control cAMP levels in cardiac myocytes.     

Human MDR1 and MRP1 Recognize Berberine as Their Transport Substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.     

Human MDR1 and MRP1 recognize berberine as their transport substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.