Search

Ions and water transported across the endothelium lining the blood–brain barrier contribute to the fluid secreted into the brain and are important in maintaining appropriate volume and ionic composition of brain interstitial fluid. Changes in this secretion process may occur after stroke. The present study identifies at transcript and protein level ion transporters involved in the movement of key ions and examines how levels of certain of these alter following oxidative stress. Immunohistochemistry provides evidence for Cl−/HCO3− exchanger, AE2, and Na+, HCO3− cotransporters, NBCe1 and NBCn1, on brain microvessels. mRNA analysis by RT-PCR reveals expression of these transporters in cultured rat brain microvascular endothelial cells (both primary and immortalized GPNT cells) and also Na+/H+ exchangers, NHE1 (primary and immortalized) and NHE2 (primary cells only). Knock-down using siRNA in immortalized GPNT cells identifies AE2 as responsible for much of the Cl−/HCO3− exchange following extracellular chloride removal and NHE1 as the transporter that accounts for most of the Na+/H+ exchange following intracellular acidification. Transcript levels of both AE2 and NHE1 are increased following hypoxia/reoxygenation. Further work is now required to determine the localization of the bicarbonate transporters to luminal or abluminal membranes of the endothelial cells as well as to identify and localize additional transport mechanisms that must exist for K+ and Cl−.

Glutathione export from uninfected human erythrocytes was compared with that from cells infected with the malaria parasite Plasmodium falciparum using two separate methods that distinguish between oxidized (GSSG) and reduced (GSH) glutathione. One involved enzymatic recycling with or without thiol-masking; the other involved rapid derivatization followed by HPLC. Glutathione efflux from uninfected erythrocytes under physiological conditions occurred predominantly as GSH. On exposure of the cells to oxidative challenge, efflux of GSSG exceeded that of GSH. Efflux of both species was blocked by MK571, an inhibitor of mammalian multidrug-resistance proteins. Glutathione efflux from parasitized erythrocytes was substantially greater than that from uninfected erythrocytes. Under physiological conditions, the exported species was GSH, whereas under energy-depleted conditions, GSSG efflux occurred. Glutathione export from parasitized cells was inhibited partially by MK571 and more so by furosemide, an inhibitor of the ‘new permeability pathways’ induced by the parasite in the host erythrocyte membrane. Efflux from isolated parasites occurred as GSH. On exposure to oxidative challenge, this GSH efflux decreased, but no GSSG export was detected. These results are consistent with the view that the parasite supplies its host erythrocyte with GSH, much of which is exported from the infected cell via parasite-induced pathways.

Multidrug efflux transporters of the ATP-Binding cassette (ABC) family, P-glycoprotein (Pgp), multidrug-resistance associated protein 4 (MRP4) and breast cancer resistance protein (BCRP), located on endothelial cells lining brain vasculature play important roles in limiting movement of substances into and enhancing their efflux from the brain. Signals from the surrounding brain normally maintain such barrier function but these may become altered in CNS pathologies such as Alzheimer's disease (AD). Previous studies have reported decreases in the glucose transporter, Glut-1, in brain vasculature of AD patients. The present study investigates the status of the multidrug efflux transporters. Sections of frozen brain from hippocampal region obtained from male AD and age-matched non-demented cases were examined for amyloid plaques and Dkk-1 expression and subjected to dual fluorescence immunochemical staining using antibodies against Pgp, BCRP or MRP4 and von Willebrand factor. Protein expression of each transporter was assessed using confocal microscopy, quantifying peak fluorescence values of cross sectional profiles across brain microvessels. Results in brain microvessels revealed expression of Pgp protein to be significantly lower in hippocampal vessels of patients with AD compared to normal individuals whereas that of MRP4 or BCRP protein was not. By contrast, analysis of the sections at protein level via Western blotting or at transcript level by qRT-PCR did not reveal significantly lower expression for either Pgp or BCRP. Such analysis did however reveal higher than normal expression in the AD brains of MRP4, probably due to gliosis, MRP4 being present also in glial cells.

Levels of multidrug efflux transporter P-glycoprotein (P-gp) on endothelial cells lining brain blood vessels are important for limiting access of many compounds to the brain. In vivo studies have indicated that ischaemia-reperfusion that generates reactive oxygen species also increases P-gp levels in brain endothelial cells. To investigate possible mechanisms, in vitro studies were performed on immortalised (GPNT) and primary rat brain endothelial cells. Exposure to hydrogen peroxide (200 microM) resulted in intracellular oxidative stress as detected from higher levels of dichlorofluorescein fluorescence and raised levels of P-gp protein, mdr1a and mdr1b transcripts and, in GPNT cells, increased mdr1a and mdr1b promoter activity. The P-gp protein increases were abolished by pre-treatment with polyethylene glycol-catalase and were curtailed by co-culture with primary rat astrocytes. Exposure of GPNT cells to 6 h hypoxia followed by 24 h reoxygenation produced less intracellular oxidative stress as judged from smaller increments in dichlorofluorescein fluorescence but still resulted in raised levels of P-gp protein, an effect partially abolished by pre-treatment with polyethylene glycol-catalase. However, transcript levels and promoter activities were not significantly increased. These data suggest that hydrogen peroxide contributes to P-gp up-regulation following hypoxia-reoxygenation but the underlying mechanisms of its actions differ from those occurring after direct hydrogen peroxide application.

Multidrug efflux transporters protect cells in the brain from potentially harmful substances but also from therapeutically useful drugs. Thus any condition that causes changes in their expression is of some importance with regard to drug access. In this study, changes in efflux transporter expression are investigated in mice containing a mutant constitutively active glycogen synthase kinase-3 (GSK-3beta) transgene, driven by the Thy-1 promoter so limiting its localization predominantly to neurons and some glial cells. As expected, decreases in beta-catenin were evident via Western blot analyses of cortical homogenates prepared from brains of these transgenic mice. As assessed by real time qRT-PCR, decreased transcript levels of the mdr1b isoform of P-glycoprotein, Mrp1 and Mrp4, (transporters associated with neurons and/or glial cells) were observed in the cortex but not the subventricular zone or hippocampus of the transgenic compared to wild type mouse brains. By contrast, no such decreases were evident with the mdr1a isoform of P-glycoprotein and Bcrp, transporters predominantly found in brain endothelium. Such transporter expression changes could not be accounted for by alterations in blood vessel density or neuronal to glial cell ratios as analyzed both from immunocytochemical staining and from RT-PCR. These observations support previous in vitro data showing that manipulations to GSK-3beta activity that alter signaling via beta-catenin can influence the expression of efflux transporters. Implications from this are that drug distribution into cells within the brain of these transgenic mice could be enhanced, hence warranting further investigation.

Fluid secretion across the blood–brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO3−, which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pHi). To analyse the transporters that may be involved in the maintenance of pHi and hence secretion of HCO3−, we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO2/HCO3−. pHi in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO3− than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO3− were similar. In the nominal absence of HCO3− the rate of acid loading was increased equally by removal of external Na+ or by inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO3− the increase in the rate of acid loading when Na+ was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na+ and HCO3−. Removal of Cl− in the presence of HCO3− produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na+ following removal of Cl− increased the rate of acid gain. In the presence of HCO3− and initial presence of Na+ and Cl−, DIDS inhibited the changes in pHi produced by removal of either Na+ or Cl−. These are the expected results if these cells possess an AE-like Cl−/HCO3− exchanger, a ‘channel-like’ permeability allowing slow influx of acid (or efflux of HCO3−), a NBC-like Cl−-independent Na+−HCO3− cotransporter, and a NHE-like Na+/H+ exchanger. The in vitro rates of HCO3− loading via the Na+−HCO3− cotransporter could, if the transporter is located on the apical, blood-facing side of the cells, account for the net secretion of HCO3− into the brain.

Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barrier sites such as the blood–brain barrier may affect distribution of steroids used for treating chronic inflammatory conditions and thus the extent to which they may perturb the hypothalamic–pituitary–adrenal axis. In the present study, six different glucocorticoids were investigated for their possible interactions with these efflux transporters. Beclomethasone dipropionate, mometasone furoate and ciclesonide active principle but not fluticasone propionate or triamcinolone, (all at 0.1 to 10 μM) caused inhibition of efflux, resulting in increased accumulation of mitoxantrone in BCRP-expressing MCF7/MR breast cancer cells and of calcein in p-glycoprotein-expressing SW620/R colon carcinoma cell. At 5 μM the same three increased sensitivity of p-glycoprotein-expressing SW620/R to doxorubicin and stimulated ATPase activity associated with BCRP expressed in bacterial membrane vesicles. Budesonide also stimulated ATPase activity. These data demonstrate the capacity of some clinically used glucocorticoids to interact with efflux transporters.

Plant flavonoids are polyphenolic compounds commonly found in vegetables, fruits and many food sources that form a significant portion of our diet. These compounds have been shown to interact with several ATP-Binding Cassette transporters that are linked with anticancer and antiviral drug resistance and as such, may be beneficial in modulating drug resistance. The present study investigates the interactions of six common polyphenols; quercetin, silymarin, resveratrol, naringenin, daidzein and hesperetin with the multidrug resistance associated proteins, MRP1, MRP4 and MRP5. At non-toxic concentrations, several of the polyphenols were able to modulate MRP1-, MRP4- and MRP5- mediated drug resistance though to varying extents. The polyphenols also reversed resistance to NSC251820, a compound that appears to be a good substrate for MRP4 as had been predicted by data mining studies. Furthermore, most of the polyphenols showed direct inhibition of MRP1-mediated [3H]-dinitrophenyl S-glutathione and MRP4-mediated [3H]-cGMP transport in inside-out vesicles prepared from human erythrocytes. Additionally, both quercetin and silymarin were found to inhibit MRP1-, MRP4-, and MRP5-mediated transport from intact cells with high affinity. They also had significant effects on ATPase activity of MRP1 and MRP4 without having any effect on [α-32P]8-azidoATP binding to these proteins. This suggests that these flavonoids most likely interact at the transporter’s substrate-binding sites. Collectively, these results suggest that dietary flavonoids such as quercetin and silymarin can modulate transport activities of MRP1, 4 and 5. Such interactions could influence bioavailability of anticancer and antiviral drugs in vivo and thus, should be considered for increasing efficacy in drug therapies.

Parasitic protozoa are responsible for a wide spectrum of diseases in humans and domestic animals. The main line of defence available against these organisms is chemotherapy. However, the application of chemotherapeutic drugs has resulted in the development of resistance mechanisms, which limit the number of antiprotozoal drugs that are effective in the treatment and control of parasitic diseases. Knowledge about the resistance mechanisms involved may allow the development of new drugs that minimise or circumvent drug resistance or may identify new targets for drug development. This review focuses on the role of protozoal ATP-binding cassette (ABC) transporters in drug resistance. These membrane proteins mediate the ATP-dependent transport of a wide variety of chemotherapeutic drugs away from their targets inside the parasites. The genome sequence of Plasmodium falciparum and Plasmodium yoelii has recently been completed, and the sequencing of other parasitic genomes are now underway. As a result, many new membrane transporters belonging to the ABC superfamily are being discovered. We review the ABC transporters in major parasitic protozoa, including Plasmodium, Leishmania, Trypanosoma and Entamoeba species. Transporters with an established role in drug resistance have been emphasised, but newly discovered transporters with a significant amino acid sequence identity to established ABC drug transporters have also been included.

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 3 micro m. This transport is reduced by MRP inhibitors and substrates including MK-571, methotrexate, estradiol 17-beta-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.

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.

The ATP-dependent transport of natural product drugs, e.g. vincristine, by multidrug resistance-associated protein (MRP1) requires reduced glutathione (GSH), whilst that of anionic substrates does not. The present results suggest, however, that GSH can modulate transport of anionic species. Efflux of fluorescent anionic substrates was measured from adherent MRP1-expressing human multidrug-resistant lung tumour cells, COR-L23/R, and drug-sensitive parental cells. As expected, much greater efflux of calcein, methylfluorescein-glutathione (GS-MF), and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was observed from the resistant cells. Unexpectedly, lowering GSH levels in COR-L23/R cells by inhibiting GSH synthesis with buthionine sulfoximine decreased efflux of calcein and of GS-MF (3-fold and 1.6-fold) but not efflux of BCECF. Transport of the anionic conjugate dinitrophenyl-glutathione ([ 3 H]DNP-SG) was investigated by following its uptake into inside-out plasma membrane vesicles prepared from the MRP1-expressing cells. At least 90% of the ATP-dependent uptake was blockable by the anti-MRP1 antibody QCRL-3 and 100 μM vincristine inhibited uptake but only in the presence of 1–3 mM GSH, suggesting MRP1 to be the protein primarily responsible for this transport. Agents shown to reduce efflux of calcein from resistant cells, i.e. indomethacin, MK-571, and probenecid, also inhibited [ 3 H]DNP-SG uptakes, consistent with MRP1 being responsible for export of calcein. At concentrations achievable within cells, GSSG (70 μM) inhibited uptake whereas GSH (1 and 3 mM) enhanced uptake. We suggest that variations in both GSH and GSSG levels within cells may affect MRP1-mediated anion transport.

Background: The ATP-dependent drug transporter proteins, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) are known to be involved in drug efflux that reduces drug accumulation and so renders tumor cells resistant to the cytotoxic effects of a number of anticancer agents. The ways in which these transporters bring about drug expulsion are not fully explained and may involve intracellular factors as well. Thus detailed evidence may be difficult to obtain from studies on intact cells.-Material and methods: Inside-out plasma membrane vesicles prepared from multidrug-resistant cells expressing high amounts of Pgp or of MRP provide a simpler system for investigating the interactions of putative substrates and resistance modifiers with the transport process. We consider here some aspects of the accumulation of radiolabelled vincristine and of dinitrophenol glutathione conjugate by these vesicles and demonstrate the usefulness of this approach for determining whether potential inhibitors have their effects on transport at the cell membrane or by more indirect means. Conclusions:We show that information gained from analysis of the ATP-dependence, time course and osmotic sensitivity of accumulation is helpful in distinguishing between transport and changes in binding. We have also used the technique to demonstrate the effects of the resistance modifier, XR-9051 on Pgp-mediated transport and to explore interactions of MK571, indomethacin and ethacrynic acid with MRP.

Iodide efflux, an index of anion permeability, has been monitored in cultured rat brain endothelial cells. Following hypotonicity-induced swelling, large, rapid increases in permeability occur, the extent of these increases depending on the degree of hypotonicity. Such large responses are not observed with rat aortic endothelial cells. Results of anion substitution experiments suggest that iodide efflux is via a chloride channel rather than an exchanger. The efflux increase is blocked by NPPB (100 microM) but not by DIDS or DPC at 100 microM. It is dependent on intracellular ATP but unaffected by removal of external calcium. Increasing internal calcium using A23187 does not produce a change in efflux, but depletion of calcium reduces or eliminates the response to hypotonicity. The response is reduced by pimozide (2-50 microM) that inhibits the actions of calmodulin and by pBPB (10 microM) that affects phospholipase A2 activity. It is eliminated by 5-lipoxygenase inhibitors (L-656,224 and ETH615, 10 microM) but is unaffected by cyclo-oxygenase inhibitors (indomethacin and piroxicam, 1-100 microM). It is blocked by some modulators of P-glycoprotein activity, e.g., verapamil (100 microM), tamoxifen (50 microM), and progesterone (100 microM) but not by others, e,g., forskolin (40 microM), dideoxyforskolin (40 microM), quinidine (100 microM) and cyclosporin A (10 microM).

Anion transport in human multidrug-resistant large cell lung tumour cells (COR-L23/R) which overexpress the multidrug-resistance-associated protein (MRP) has been compared with that in cells of the parent line (COR-L23/P). Whole-cell patch-clamp recordings reveal variability between individual cells in basal anion conductance and in anion conductance increases following exposure to hypotonic media. The increase of stimulated over basal conductance is significantly larger for resistant cells than for parent cells. The chloride channel blocker, diisothiocyanatostilbene-2-2'-disulphonic acid (DIDS), rapidly and reversibly inhibits the increase in outward but not inward conductance when applied externally at 10(-4) M during recording, but it is without effect when introduced into the cells via the patch pipette. Preincubation with DIDS greatly reduces both inward and outward conductance. 125I- efflux has been used to measure anion movement in cell populations. Basal efflux is similar in the two cell lines, but following a hypotonic challenge, the increase in rate constant for efflux from COR-L23/R cells is at least double that from COR-L23/P cells. This increase in efflux is greatly reduced by incubation with DIDS at 10(-4) M. Replacement of external chloride by gluconate does not affect efflux, thus excluding the possible involvement of DIDS-sensitive chloride exchange. Results from both techniques suggest that DIDS-sensitive, hypotonicity-induced anion channel activity is augmented in COR-L23/R multidrug-resistant variant cells which overexpress MRP. This augmentation may be caused by MRP itself or by other genes coexpressed with MRP.

Ischemia-reperfusion leads to increased levels at the blood-brain barrier of the multidrug efflux transporter, P-glycoprotein that provides protection to the brain by limiting access of unwanted substances. This is coincident with the production of nitric oxide. This present study using immortalized rat brain endothelial cells (GPNTs) examines whether following hypoxia-reoxygenation, nitric oxide contributes to the alterations in P-glycoprotein levels. After 6 h of hypoxia, both nitric oxide and reactive oxygen species, detected intracellularly using fluorescent monitoring dyes, were produced in the subsequent reoxygenation phase coincident with increased P-glycoprotein. The evidence that nitric oxide can directly affect P-glycoprotein expression was sought by applying S-nitroso-N-acetyl-DL: -penicillamine that as shown increased the nitric oxide generation. Sodium nitroprusside, though more effective at increasing P-glycoprotein expression, appeared to produce different reactive species. Real time RT-PCR analysis revealed the predominant form of nitric oxide synthase in these cells to be endothelial, inhibition of which partially prevented the increase in P-glycoprotein during reoxygenation. These data indicate that the production of nitric oxide by endothelial nitric oxide synthase during reoxygenation can influence P-glycoprotein expression in cells of the blood-rat brain barrier, highlighting another route by which nitric oxide may protect the brain.

Transport activities involved in intracellular pH (pHi) recovery after acid or alkali challenge were investigated in cultured rat brain microvascular endothelial cells by monitoring pHi using a pH-sensitive dye. Following relatively small acid loads with pHi ∼ 6.5, \({\text{HCO}}_3^ - \) influx accounted for most of the acid extrusion from the cell with both Cl−-independent and Cl−-dependent, Na+-dependent transporters involved. The Cl−-independent component has the same properties as the NBC-like transporter previously shown to account for most of the acid extrusion near the resting pHi. Following large acid loads with pHi < 6.5, most of the acid extrusion was mediated by Na+/H+ exchange, the rate of which was steeply dependent on pHi. Concanamycin A, an inhibitor of V-type ATPase, had no effect on the rates of acid extrusion. Following an alkali challenge, the major component of the acid loading leading to recovery of pHi occurred by \({{{\text{Cl}}^ - } \mathord{\left/ {\vphantom {{{\text{Cl}}^ - } {{\text{HCO}}_3^ - }}} \right. \kern-\nulldelimiterspace} {{\text{HCO}}_3^ - }}\) exchange. This exchange had the same properties as the AE-like transporter previously identified as a major acid loader near resting pHi. These acid-loading and acid-extruding transport mechanisms together with the Na+, K+, ATPase may be sufficient to account not only for pHi regulation in brain endothelial cells but also for the net secretion of \({\text{HCO}}_3^ - \) across the blood–brain barrier.

This study explores the effects of neural precursor cells (NPCs) on barrier characteristics in brain vasculature. Primary rat brain endothelial cells were exposed to conditioned medium from NPCs isolated from day 14 embryonic rat brains and maintained as free-floating undifferentiated neurospheres. Such exposure increased brain endothelial transcript levels of the mdr1a but not mdr1b gene encoding P-glycoprotein (Pgp) and reduced proliferation but did not alter transendothelial resistance (TER). These effects were compared to those seen following co-culture with differentiating NPCs or with primary astrocytes. NPCs, if grown adherent, differentiate into glial and neuronal cells as assessed by immunocytochemical and mRNA analysis. Brain endothelial cells when co-cultured with these cells also showed reduced proliferation and enhanced mdr1a expression, but in addition increased TER. Similar increases were observed in co-culture with astrocytes. These results suggest that undifferentiated NPCs produce factors that influence Pgp expression whereas their progeny also affect tight junction integrity.

This study investigated mRNA expression and protein localization of equilibrative and concentrative nucleoside transporters (ENTs, CNTs) in primary cultures of rat brain endothelial cells (RBEC) and rat choroid plexus epithelial cells (RCPEC). Reverse transcriptase PCR analysis revealed that RBEC and RCPEC contained mRNA for rENT1, rENT2 and rCNT2 and for rENT1, rENT2, rCNT2 and rCNT3, respectively. Immunoblotting of membrane fractions of RBEC, fresh RCPEC and primary cultures of RCPEC revealed the presence of rENT1, rENT2 and rCNT2 proteins in all samples. Measurement of [14C]adenosine uptake into cells grown as monolayers on permeable plastic supports revealed a polarized distribution of Na+-dependent adenosine uptake in that CNT activity was associated exclusively in membranes of RBEC facing the lower chamber (which corresponds to the surface facing the interstitial fluid in situ) and in membranes of RCPEC facing the upper chamber (which corresponds to the surface facing the cerebrospinal fluid in situ). In both RBEC and RCPEC, adenosine uptake from the opposite chambers was Na+-independent and partially inhibited by nitrobenzylthioinosine, indicating the presence of the equilibrative sensitive transporter rENT1.

Human erythrocytes produce cGMP that can be eliminated by phosphodiesterases or active efflux transporters. The efflux can be studied under controlled conditions as ATP-dependent uptake into inside-out membrane vesicles. However, widely differing values for the transport rates have been reported. We have here examined factors that influence the uptake rates measured and thus may explain these discrepancies. Both the ionic composition of the buffer used during uptake and the mode of vesicle preparation were found to affect the observed transport rates. Furthermore it was apparent that different blood donors expressed on their erythrocytes different amounts of both MRP4 and MRP5, transporters that have been putatively linked to cGMP efflux across erythrocyte membranes. These differences in expression were reflected in differences in rates of cGMP uptake into inside-out erythrocyte membrane vesicles. Calculations based on the transport rates observed using vesicles suggest that efflux may be the principal means for eliminating cGMP from human erythrocytes.

Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development. Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs). A gene, PFA0590w, encoding a MRP homologue, has been identified in P. falciparum. Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting. A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210–215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein. Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole. We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.

Multidrug transporters influence drug distribution in vivo and are often associated with tumour drug resistance. Here we show that plant-derived polyphenols that interact with P-glycoprotein can also modulate the activity of the recently discovered ABC transporter, breast cancer resistance protein (BCRP/ABCG2). In two separate BCRP-overexpressing cell lines, accumulation of the established BCRP substrates mitoxantrone and bodipy-FL-prazosin was significantly increased by the flavonoids silymarin, hesperetin, quercetin, and daidzein, and the stilbene resveratrol (each at 30 microM) as measured by flow cytometry, though there was no corresponding increase in the respective wild-type cell lines. These compounds also stimulated the vanadate-inhibitable ATPase activity in membranes prepared from bacteria (Lactococcus lactis) expressing BCRP. Given the high dietary intake of polyphenols, such interactions with BCRP, particularly in the intestines, may have important consequences in vivo for the distribution of these compounds as well as other BCRP substrates.