ATP-dependent Uptake Research Articles

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca2+ concentration ([Ca2+]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca2+]i. Chelating Ca2+ ions in the extracellular medium suppressed the intracellular Ca2+ signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca2+- and P2X7-independent transport mechanism in macrophages.

Functional coupling of chloride–proton exchanger ClC-5 to gastric H+,K+-ATPase

SummaryIt has been reported that chloride–proton exchanger ClC-5 and vacuolar-type H+-ATPase are essential for endosomal acidification in the renal proximal cells. Here, we found that ClC-5 is expressed in the gastric parietal cells which secrete actively hydrochloric acid at the luminal region of the gland, and that it is partially localized in the intracellular tubulovesicles in which gastric H+,K+-ATPase is abundantly expressed. ClC-5 was co-immunoprecipitated with H+,K+-ATPase in the lysate of tubulovesicles. The ATP-dependent uptake of 36Cl− into the vesicles was abolished by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH28080), an inhibitor of H+,K+-ATPase, suggesting functional expression of ClC-5. In the tetracycline-regulated expression system of ClC-5 in the HEK293 cells stably expressing gastric H+,K+-ATPase, ClC-5 was co-immunoprecipitated with H+,K+-ATPase, but not with endogenous Na+,K+-ATPase. The SCH28080-sensitive 36Cl− transporting activity was observed in the ClC-5-expressing cells, but not in the ClC-5-non-expressing cells. The mutant (E211A-ClC-5), which has no H+ transport activity, did not show the SCH28080-sensitive 36Cl− transport. On the other hand, both ClC-5 and its mutant (E211A) significantly increased the activity of H+,K+-ATPase. Our results suggest that ClC-5 and H+,K+-ATPase are functionally associated and that they may contribute to gastric acid secretion.

Transplacental transfer and distribution of pravastatin

The objective of the study was to determine the bidirectional transfer of pravastatin across the dually perfused term human placental lobule and its distribution between the tissue and maternal and fetal circuits. The transfer of pravastatin was determined in the maternal-to-fetal (n= 11) and fetal-to-maternal (n= 10) directions. Pravastatin was coperfused with its [(3)H]-isotope and the marker compound antipyrine (20 μg/mL) and its [(14)C]-isotope. The concentration of pravastatin in the perfused tissue and the maternal and fetal circuits was determined using liquid scintillation spectrometry. Inside-out vesicles prepared from placental brush border membranes were utilized to investigate the role of efflux transporters in the transplacental transfer of pravastatin. Pravastatin was transferred from the maternal to the fetal circuit and vice versa. In the maternal-to-fetal direction, the distribution of pravastatin at the end of experiment was as follows: 14 ± 5% of the drug was retained by the tissue, 68 ± 5% remained in the maternal circuit, and 18 ± 4% was transferred to the fetal circuit. Thenormalized transfer of pravastatin (clearance index) to antipyrine in the fetal-to-maternal direction (0.48 ± 0.07) was higher than its transfer in the maternal-to-fetal direction (0.36 ± 0.07, P < .01). Furthermore, pravastatin inhibited the adenosine triphosphate (ATP)-dependent uptake of the paclitaxel and estrone sulfate. The transfer of pravastatin across the dually perfused placental lobule suggests that fetal exposure to pravastatin is plausible. The higher transfer of pravastatin in the fetal-to-maternal direction than the reverse as well as its inhibition of the ATP-dependent uptake of [(3)H]-paclitaxel and [(3)H]-estrone sulfate strongly suggest the involvement of efflux transporters in decreasing its transfer across the placenta and support pravastatin's favorable pharmacokinetic profile in pregnancy.

A membrane vesicle-based assay to enable prediction of human biliary excretion

1. Prediction of biliary excretion is a challenge due to the lack of in vitro assays. Our laboratory previously demonstrated a highly significant correlation between in vitro IC50 values against mrp2 using rat canalicular liver plasma membrane vesicles and in vivo biliary excretion (Colombo et al., 2012). This study explores the possibility of predicting in vivo biliary excretion in human using membrane vesicles prepared from MDCKII cells transfected with human ABCC2.2. In vitro MRP2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) in inside-out membrane vesicles isolated from MDCK-ABCC2 cells. CDCF uptake was time- and concentration-dependent (Km of 4.0 ± 1.2 µM and a Vmax of 7.8 ± 0.9 pmol/mg/min) and inhibited by benzbromarone and MK-571 with IC50 values of 1.2 and 7.6 µM, respectively.3. A significant linear correlation (r2 = 0.790) between the in vitro IC50 values from the described MRP2 assay and in vivo biliary excretion in humans was observed using 11 well-documented drugs covering low to high biliary excretions.4. This study demonstrates, for the first time, that inhibition of CDCF uptake in MDCKII-ABCC2 vesicles not only provides a screening assay to assess MRP2 drug–drug interaction potential, but is also predictive of human MRP2-mediated biliary excretion.

Substrate translocation and stimulated ATP hydrolysis of human ABC transporter MRP3 show positive cooperativity and are half-coupled

ABC transporters are involved in countless processes from lipid excretion over cellular detoxification to multidrug resistance of cancer cells. The latter is especially conferred by the ABCC subfamily also called multidrug resistance-associated proteins (MRPs) that excrete a variety of amphipathics including anticancer drugs by ATP-dependent transport. As the mechanisms of substrate translocation and ATP hydrolysis are still unclear for MRPs, we investigated the kinetics of both processes with focus on cooperativity and coupling between ATPase activity and substrate transport using purified MRP3 in proteoliposomes. Although the ATP-dependent uptake of amphipathics and the hydrophilic 5(6)-carboxy-2′-7′-dichlorofluorescein (CDCF) into the lumen of proteoliposomes showed affinity constants similar to those reported for cell-based assays, the maximal uptake rates were up to 250 times higher. Moreover, all substrates showed cooperative interactions of two subunits. Upon stimulation with amphipathics, ATPase activity of MRP3 increased from 80nmol/(mgmin) to 180nmol/(mgmin) showing positive cooperativity with a Hill coefficient of 2. While Hill coefficient and maximal ATPase activity were found to be substrate independent, the affinity constants are characteristic for a given substrate and correspond to the value for transport. Therefore, cooperative interactions of the two nucleotide binding domains (NBDs) in MRP3 are mediated by substrate binding to the transmembrane domains (TMDs). In contrast to amphipathic substrates, CDCF did not stimulate ATPase activity despite being transported in an ATP-dependent manner. This indicates that ATP hydrolysis and substrate translocation are half-coupled in MRP3 as CDCF shuttles on a basal TMD activity resulting from the basal ATPase activity.

Proapoptotic miltefosine nanovesicles show synergism with paclitaxel: Implications for glioblastoma multiforme therapy

Hexadecylphosphocholine (HePC) or miltefosine based proapoptotic lipid nanovesicles encapsulating paclitaxel for synergistic anticancer effect of paclitaxel and miltefosine in chemoresistant human glioblastoma multiforme (U-87 MG) overexpressing multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp), were developed in this study. The nanovesicles had 100–200nm size and a negative zeta potential (∼−25mV) and optimized for miltefosine content based on their physiochemical properties. With a high encapsulation efficiency of 94%, the nanovesicles showed sustained release of paclitaxel in nasal fluid and triggered release in the cerebrospinal fluid. Synergistic action of paclitaxel and miltefosine was observed with a low IC50 of 162±5nM. The nanovesicle also overcame drug resistance and showed ATP dependent uptake via clathrin mediated pathway in glioblastoma cells. An improved therapeutic efficacy in comparison to Taxol®, the current clinical formulation of paclitaxel was observed. Efficient brain uptake of the nanovesicles upon intranasal administration was observed in vivo and the nanovesicles were also found to be able to cross blood brain barrier. These studies therefore suggest the therapeutic potential of proapoptotic lipid nanovesicles and their feasibility for intranasal administration in the treatment of chemoresistant glioblastoma.

Multi trigger responsive, surface active lipid nanovesicle aerosols for improved efficacy of paclitaxel in lung cancer

The present study focuses on the development of multi-trigger responsive surface active lipid nanovesicles encapsulating paclitaxel with the hypothesis that pulmonary surfactant mimetic lipid vesicles sensitive to temperature and enzyme simultaneously will offer synergistic advantage towards improved therapeutic efficacy of paclitaxel via aerosol administration. The nanovesicles showed a unimodal size distribution of the particles (100-150 nm) and high encapsulation efficiency of paclitaxel (82%). Triggered release of paclitaxel was observed at ∼42 °C in the presence of secretory phospholipase A(2) enzyme with maximum release observed with both the triggers used simultaneously. Since these nanovesicles are intended for aerosol administration in the treatment of lung cancer, they were engineered to have high surface activity and airway patency, in order to mimic pulmonary surfactant functions. High deposition of nanovesicles in the lower impingement chamber of a twin impinger upon nebulization suggested them to be capable of reaching the terminal regions of the lungs. Nanovesicles showed facilitated and ATP dependent active uptake by A549 cells. The cytotoxic potential of the nanovesicles was significantly increased upon simultaneous use of both the triggers with an IC(50) of 49.3 nM. Overall, these studies suggest the therapeutic potential and advantages of multi trigger responsive lipid nanovesicles with encapsulated paclitaxel over that of the commercially available form of paclitaxel namely Taxol, and suggests the feasibility of aerosol administration in the treatment of lung cancer and pulmonary metastasis.

Effects of glycyrrhizin on biliary transport and hepatic levels of glutathione in rats

The purpose of the current study was to determine whether glycyrrhizin (GL) maintains hepatic glutathione (GSH) levels by inhibiting GSH biliary secretion in normal rats. The effects of glycyrrhizin on hepatic glutathione content, bile flow and biliary secretion of glutathione were examined. Because glutathione is a substrate for multidrug resistance associated protein-2 (Mrp2/ABCC2), the inhibitory effects of GL on Mrp2 in isolated perfused rat liver and in Mrp2-expressing Sf9 membrane vesicles were also examined using the Mrp2 substrate methotrexate (MTX) and estradiol-17-β-glucuronide (E2 17G). The hepatic content of glutathione in rats following GL perfusion (43.7 µmol/l) in isolated liver perfusion and GL intravenous treatment (25 mg/kg) was significantly higher than that for the control. A marked and dose-dependent decrease in the excretion of glutathione was observed. In addition, the secretion rate of MTX was decreased by 57% in isolated liver perfusion in GL-treated rats. Moreover the ATP-dependent uptake of E2 17G by Mrp2 membrane vesicles was decreased by 75.9% in the 20 µm GL group and by 60.5% in the 2 µm GL group. In conclusion, glycyrrhizin increases hepatic glutathione content possibly through inhibition of Mrp2 which then reduces the biliary excretion of glutathione.

Association of multidrug resistance-associated protein 2 single nucleotide polymorphism rs12762549 with the basal plasma levels of phase II metabolites of isoflavonoids in healthy Japanese individuals

Multidrug resistance-associated protein 2 (MRP2; ABCC2) is an ATP-binding cassette transporter that mediates the efflux of anionic drugs and phase II metabolites. Our aim was to elucidate the impact of a single nucleotide polymorphism, rs12762549 (G>C), on the in-vivo activity of MRP2. Plasma specimens collected from 18 healthy volunteers were subjected to an untargeted metabolomic analysis using liquid chromatography-mass spectrometry. The role of MRP2 in the disposition of substances of interest was then examined in vivo in Mrp2-deficient mutant rats (Eisai hyperbilirubinemic rats; EHBRs) and in vitro in human MRP2-expressing membrane vesicles. A multivariate analysis model using liquid chromatography-mass spectrometry data sets successfully differentiated the GG and the CC genotypes of rs12762549. The C allele is associated with the basal plasma levels of the five phase II metabolites of genistein and dihydrogenistein. Such phase II metabolites were also accumulated in EHBR, compared with normal rats, partly because of the reduced biliary excretion. Following oral administration of genistein and daidzein, the plasma concentrations of total genistein and total daidzein, which were mainly accounted for by sulfoglucuronide conjugates, were markedly higher in EHBR than in normal rats. ATP-dependent uptake of sulfoglucuronides and glucuronides of the isoflavonoids was observed only in MRP2-expressing membrane vesicles. MRP2 plays a significant role in preventing the accumulation of phase II metabolites of isoflavonoids. The rs12762549 is associated with an interindividual difference in the plasma levels of MRP2 substrates, phase II metabolites of isoflavonoids, suggesting that this single nucleotide polymorphism is associated with variations in in-vivo MRP2 activity.

A high throughput in vitro mrp2 assay to predict in vivo biliary excretion

Prediction of biliary excretion is a challenge for drug discovery scientists due to the lack of in vitro assays. This study explores the possibility of establishing a simple assay to predict in vivo biliary excretion via the mrp2 transport system.In vitro mrp2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) in canalicular plasma membrane vesicles (cLPM) from rat livers. The CDCF uptake was time- and concentration-dependent (Km of 2.2 ± 0.3 µM and Vmax of 115 ± 26 pmol/mg/min) and strongly inhibited by the mrp2 inhibitors, benzbromarone, MK-571, and cyclosporine A, with IC50 values ≤ 1.1 µM.Low inhibition of CDCF uptake by taurocholate (BSEP inhibitor; 57 µM) and digoxin (P-gp inhibitor; 101 µM) demonstrated assay specificity towards mrp2.A highly significant correlation (r2 = 0.959) between the in vitro IC50 values from the described mrp2 assay and in vivo biliary excretion in rats was observed using 10 literature compounds.This study demonstrated, for the first time, that a high throughput assay could be established with the capability of predicting biliary excretion in the rat using CDCF as a substrate.

Proapoptotic lipid nanovesicles: Synergism with paclitaxel in human lung adenocarcinoma A549 cells

The present study focuses on the development and evaluation of phosphatidylserine based proapoptotic lipid nanovesicles (PSN-PTX) as aerosols for synergistic activity with paclitaxel against lung cancer. PSN-PTX showed a unimodal size distribution of the particles (100–200 nm), negative surface charge of − 29 mV and high encapsulation efficiency of paclitaxel (82%) with 19% of it releasing in 48 h. PSN-PTX was found to be highly surface active as compared to Taxol®, marketed formulation of paclitaxel, whose surface activity was found to be detrimental for pulmonary mechanics. PSN-PTX also showed high airway patency in capillary surfactometer unlike Taxol®, suggesting its ability to mimic pulmonary surfactant functions. High deposition of PSN-PTX in lower impingement chamber of twin impinger upon nebulization suggested it to be capable of reaching the terminal regions of the lungs. Nanovesicles showed facilitated and ATP dependent active uptake by A549 cells. The combination of phosphatidylserine nanovesicles and paclitaxel as PSN-PTX enhanced cytotoxicity in A549 cell line showing an IC 50 of 18 nM which is10-50 folds less than the IC 50 values observed for blank phosphtidylserine nanovesicles and paclitaxel alone. Further, the combination index was found to be less than one which indicates a synergism of the two components. DNA fragmentation study showed that blank phosphatidylserine nanovesicles induce apoptosis in A549 cells and hence behave as proapoptotic nanovesicles in the combination therapy. Overall, these studies suggest the therapeutic potential and advantages of combination chemotherapy of proapoptotic lipid nanovesicles with encapsulated paclitaxel and their feasibility for aerosol administration in the treatment of lung cancer.

Expression and Function of Human MRP1 (ABCC1) Is Dependent on Amino Acids in Cytoplasmic Loop 5 and Its Interface with Nucleotide Binding Domain 2

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.

Glycosomal ABC transporters of Trypanosoma brucei: Characterisation of their expression, topology and substrate specificity

Metabolism in trypanosomatids is compartmentalised with major pathways, notably glycolysis, present in peroxisome-like organelles called glycosomes. To date, little information is available about the transport of metabolites through the glycosomal membrane. Previously, three ATP-binding cassette (ABC) transporters, called GAT1–3 for Glycosomal ABC Transporters 1 to 3, have been identified in the glycosomal membrane of Trypanosoma brucei. Here we report that GAT1 and GAT3 are expressed both in bloodstream and procyclic form trypanosomes, whereas GAT2 is mainly or exclusively expressed in bloodstream-form cells. Protease protection experiments showed that the nucleotide-binding domain of GAT1 and GAT3 is exposed to the cytosol, indicating that these transporters mediate the ATP-dependent uptake of solutes from the cytosol into the glycosomal lumen. Depletion of GAT1 and GAT3 by RNA interference in procyclic cells grown in glucose-containing medium did not affect growth. Surprisingly, GAT1 depletion enhanced the expression of the very different GAT3 protein. Expression knockdown of GAT1, but not GAT3, in procyclic cells cultured in glucose-free medium was lethal. Depletion of GAT1 in glucose-grown procyclic cells caused a modification of the total cellular fatty-acid composition. No or only minor changes were observed in the levels of most fatty acids, including oleate (C18:1), nevertheless the linoleate (C18:2) abundance was significantly increased upon GAT1 silencing. Furthermore, glycosomes purified from procyclic wild-type cells incorporate oleoyl-CoA in a concentration- and ATP-dependent manner, whilst this incorporation was severely reduced in glycosomes from cells in which GAT1 levels had been decreased. Together, these results strongly suggest that GAT1 serves to transport primarily oleoyl-CoA, but possibly also other fatty acids, from the cytosol into the glycosomal lumen and that its depletion results in a cellular linoleate accumulation, probably due to the presence of an active oleate desaturase. The role of intraglycosomal oleoyl-CoA and its essentiality when the trypanosomes are grown in the absence of glucose, are discussed.

Positron Emission Tomography Studies Using (15R)-16-m-[11C]tolyl-17,18,19,20-tetranorisocarbacyclin Methyl Ester for the Evaluation of Hepatobiliary Transport

A quantitative positron emission tomography (PET) methodology was developed for in vivo kinetic analysis of hepatobiliary transport. Serial abdominal PET scans were performed on normal and multidrug resistance-associated protein 2 (Mrp2)-deficient rats after intravenous injection of (15R)-16-m-[(11)C]tolyl-17,18,19,20-tetranorisocarbacyclin methyl ester (15R-[(11)C] TIC-Me) as a radiotracer. 15R-[(11)C]TIC-Me was rapidly converted to its acid form in blood within 10 s. PET scans revealed that 15R-[(11)C]TIC was localized mainly in the liver within 5 min of injection. By 90 min, total radioactivity in bile of Mrp2-deficient rats was significantly reduced compared with controls. Metabolite analysis by thin-layer chromatography autoradiography showed that 15R-[(11)C]TIC is converted to at least three metabolites (M1, M2, and M3), and M2 and M3 are the major metabolites in plasma and bile, respectively. Hepatic uptake clearance of total radioactivity in normal rats was close to the hepatic blood flow rate and slightly higher than that in Mrp2-deficient rats. The intrinsic canalicular efflux clearance of M3 (CL(int,bile,M3)) in Mrp2-deficient rats was decreased to 12% of controls, whereas clearance of M2 was moderately decreased (54%). An in vitro transport assay detected ATP-dependent uptake of both M2 and M3 by rat Mrp2-expressing membrane vesicles. These results demonstrated that M3 is excreted primarily into the bile by Mrp2 in normal rats. We conclude that PET studies using 15R-[(11)C]TIC-Me could be useful for in vivo analyses of Mrp2-mediated hepatobiliary transport.

Transporter Studies with the 3-O-Sulfate Conjugate of 17α-Ethinylestradiol: Assessment of Human Liver Drug Transporters

17alpha-Ethinylestradiol (EE2), a component of oral contraceptives, is known to undergo considerable first-pass 3-O-sulfation in the intestine and liver. Once formed, the 3-O-sulfate conjugate (EE2-Sul) is detected in circulation at appreciable levels (versus parent EE2) and is present in bile. Therefore, hepatic uptake of EE2-Sul was assessed with suspensions of cryopreserved human primary hepatocytes. In this instance, there was evidence for active (temperature-dependent) uptake, which was described by a two-K(m) (Michaelis constant) model (K(m1) = 220 nM; K(m2) = 15.5 microM). Uptake was inhibited (approximately 90%) by bromosulfophthalein but not by tetraethylammonium or p-aminohippurate. In agreement, EE2-Sul was shown to be a substrate of recombinant organic anion transporter peptides (OATP1B1 and OATP2B1), and Na(+)/taurocholate-cotransporting polypeptide (NTCP), expressed individually in human embryonic kidney (HEK) 293 cells. Transport by OATP1B1 was described by two K(m) values (87 nM and 141 microM), whereas OATP2B1- and NTCP-mediated uptake into HEK-293 cells conformed to single K(m) kinetics (10.7 and 2.6 microM, respectively). EE2-Sul was also assessed as an efflux transporter substrate using membrane vesicles expressing bile salt export pump, breast cancer resistance protein (BCRP), and individual forms of multidrug resistance-associated protein (MRP1, MRP2, and MRP3). Transport studies were also conducted with a cell line expression P-glycoprotein. Only vesicles that contained BCRP exhibited ATP-dependent uptake of EE2-Sul (K(m1) = 2.9 and K(m2) = 307 microM). Collectively, the data show that hepatic uptake of EE2-Sul can be mediated by three transporters (OATP1B1, OATP2B1, and NTCP), whereas biliary excretion of EE2-Sul into bile likely involves BCRP.

Effect of HEPES Buffer on the Uptake and Transport of P-Glycoprotein Substrates and Large Neutral Amino Acids

HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the growth and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of P(appB-A) to P(appA-B) in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged, glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake; elevation of cyclosporine uptake in the presence of 5 muM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories.

Involvement of a Novel ABC Transporter and Monoalkyl Phthalate Ester Hydrolase in Phthalate Ester Catabolism by Rhodococcus jostii RHA1

Phthalate esters (PEs) are important environmental pollutants. While the biodegradation of the parent compound, phthalate (PTH), is well characterized, the biodegradation of PEs is not well understood. In particular, prior to this study, genes involved in the uptake and hydrolysis of these compounds were not conclusively identified. We found that Rhodococcus jostii RHA1 could grow on a variety of monoalkyl PEs, including methyl, butyl, hexyl, and 2-ethylhexyl PTHs. Strain RHA1 could not grow on most dialkyl PEs, but suspensions of cells grown on PTH transformed dimethyl, diethyl, dipropyl, dibutyl, dihexyl and di-(2-ethylhexyl) PTHs. The major products of these dialkyl PEs were PTH and the corresponding monoalkyl PEs, and minor products resulted from the shortening of the alkyl side chains. RHA1 exhibited an inducible, ATP-dependent uptake system for PTH with a K(m) of 22 microM. The deletion and complementation of the patB gene demonstrated that the ATP-binding cassette (ABC) transporter encoded by patDABC is required for the uptake of PTH and monoalkyl PEs by RHA1. The hydrolase encoded by patE of RHA1 was expressed in Escherichia coli. PatE specifically hydrolyzed monoalkyl PEs to PTH but did not transform dialkyl PEs or other aromatic esters. This investigation of RHA1 elucidates key processes that are consistent with the environmental fate of PEs.

Chloroquine Transport inPlasmodium falciparum. 2. Analysis of PfCRT-Mediated Drug Transport Using Proteoliposomes and a Fluorescent Chloroquine Probe

Mutations in the PfCRT protein cause chloroquine resistance (CQR), and earlier studies from our laboratory using plasma membrane inside-out vesicles (ISOV) prepared from yeast expressing recombinant PfCRT [Zhang, H., et al. (2004) Biochemistry 43, 8290-8296] suggested that the putative transporter mediates downhill facilitated diffusion of charged chloroquine (CQ). However, more recent experiments with a fluorescent CQ probe (NBD-CQ) presented in the accompanying paper (DOI 10.1021/bi901034r ) indicated that the CQR phenotype in live parasites is associated with a reduced rate of ATP-dependent CQ uptake into the digestive vacuole (DV). An altered rate constant for uptake has multiple interpretations. To further investigate this phenomenon, PfCRT proteins found in chloroquine-sensitive (CQS) and CQR strains of Plasmodium falciparum were purified from yeast engineered to express "yeast optimized" pfcrt genes, reconstituted into proteoliposomes (PL), and efflux of NBD-CQ was measured from these PL. A membrane-impermeant quencher was used to distinguish intra-PL NBD-CQ from extra-PL NBD-CQ vs time as well as resolve initial rates and rate constants for efflux. Efflux was investigated at a range of NBD-CQ concentrations, in the presence vs absence of pH gradients (DeltapH) and transmembrane potentials (DeltaPsi). Explicit turnover numbers for apparent PfCRT-mediated transport were then calculated under these conditions. Our data are consistent with a model wherein PfCRT catalyzes electrochemically downhill diffusion of NBD-CQ out of the DV, in response to DeltaPsi or DeltapH, at a rate that can partially compete with the ATP-dependent uptake of NBD-CQ by CQS parasites described in the previous paper. These data allow us to propose a refined model for altered CQ accumulation in CQR malarial parasites.

249 Biology of mesothelioma

Members of the ATP-binding cassette (ABC) transporters including P-glycoprotein (Pgp/ABCB1), multidrug resistance proteins (MRPs/ABCC) as well as breast cancer resistance protein (BCRP/ABCG2) function as ATP-dependent drug efflux transporters, which form a unique defense network against multiple chemotherapeutic drugs and cellular toxins. Among antitumor agents is the important group of folic acid antimetabolites known as antifolates. Antifolates such as methotrexate (MTX), pemetrexed and raltitrexed exert their cytotoxic activity via potent inhibition of folate-dependent enzymes essential for purine and pyrimidine nucleotide biosynthesis and thereby block DNA replication. Overexpression of MRPs and BCRP confers resistance upon malignant cells to various hydrophilic and lipophilic antifolates. Apart from their central role in mediating resistance to antifolates and other anticancer drugs, MRPs and BCRP have been recently shown to transport naturally occurring reduced folates. This was inferred from various complementary systems as follows: (a) Cell-free systems including ATP-dependent uptake of radiolabeled folate/MTX into purified inside-out membrane vesicles from stable transfectants and/or cells overexpressing these transporters, (b) Decreased accumulation of radiolabeled folate/MTX in cultured tumor cells overexpressing these transporters, as well as (c) In vivo rodent models such as Eisi hyperbillirubinemic rats (EHBR) that hereditarily lack MRP2 in their canalicular membrane and thereby display a bile that is highly deficient in various reduced folate cofactors and MTX, when compared with wild type Sprague–Dawley (SD) rats. In all cases, these folate/antifolate transporters functioned as high capacity, low affinity ATP-driven exporters. While the mechanism of cellular retention of (anti)folates is mediated via (anti)folylpolyglutamylation, certain efflux transporters including MRP5 (ABCC5) and BCRP were shown to transport both mono-, di- as well as triglutamate derivatives of MTX and folic acid. Furthermore, overexpression of MRPs and BCRP has been shown to result in decreased cellular folate pools, whereas loss of ABC transporter expression brought about a significant expansion in the intracellular reduced folate pool. The latter finding has important implications to antifolate-based chemotherapy as an augmented cellular folate pool results in a significant level of resistance to certain antifolates. Hence, the aims of the present review are: (a) To summarize and discuss the cumulative evidence supporting a functional role for various multidrug resistance efflux transporters of the ABC superfamily which mediate resistance to hydrophilic and lipophilic antifolates, (b) To describe and evaluate the recent data suggesting a role for these efflux transporters in regulation of cellular folate homeostasis under folate replete and deplete conditions. Furthermore, novel developments and future perspectives regarding the identification of novel antifolate target proteins and mechanisms of action, as well as rationally designed emerging drug combinations containing antifolates along with receptor tyrosine kinase inhibitors are being discussed.

Characterization of the ATP-dependent Sphingosine 1-Phosphate Transporter in Rat Erythrocytes

Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5'-(beta,gamma-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent K(m) value of 21 microm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter.