Progress in the Molecular Characterization of Hepatobiliary Transporters.

Over the last 25 years, our understanding of the driving forces for hepatobiliary elimination and knowledge of the molecular basis of uptake and efflux transport in hepatocytes have undergone fundamental changes. This refers to bile acids and many other endogenous substances as well as to drugs that are eliminated on the hepatobiliary route. In this development, not only molecular cloning, functional characterization, and localization of transporters were decisive, but also the discovery of hereditary mutations in genes encoding sinusoidal uptake transporters and canalicular export pumps in humans and rodents. Uptake by passive diffusion and elimination into bile driven by the electrochemical gradient are no longer considered relevant for hepatobiliary elimination in the intact organism. Furthermore, insights into the relative roles of uptake transporters and unidirectional ATP-driven efflux pumps were obtained when we established double-transfected polarized cell lines stably expressing, as an example, the hepatocellular uptake transporter OATP1B3 and the apical (canalicular) efflux pump multidrug resistance protein 2 (MRP2; ABCC2). ATP-dependent efflux transporters localized to the basolateral (sinusoidal) hepatocyte membrane, particularly MRP3 (ABCC3) and MRP4 (ABCC4), pump substances from hepatocytes into sinusoidal blood. Bile acids are substrates for human MRP4 in the presence of physiological concentrations of reduced glutathione, which undergoes co-transport. These efflux pumps have been recognized in recent years to play an important compensatory role in cholestasis and to contribute to the balance between uptake and efflux of bile acids and other organic anions during the vectorial transport from blood into bile. This sinusoidal efflux not only enables subsequent renal elimination but also facilitates the re-uptake of substances into neighboring hepatocytes located more centrally and downstream in the sinusoid.

The roles of MRP2, MRP3, OATP1B1, and OATP1B3 in conjugated hyperbilirubinemia.

Increased concentrations of bilirubin glucuronides in blood plasma indicate hepatocellular dysfunction. Elucidation of the transport processes of bilirubin conjugates across the basolateral (sinusoidal) and the canalicular plasma membrane domains of hepatocytes has decisively contributed to our current understanding of the molecular basis of conjugated hyperbilirubinemia in human liver diseases. Under normal conditions, unconjugated bilirubin is taken up into hepatocytes by transporters of the organic anion-transporting polypeptide (OATP) family, followed by conjugation with glucuronic acid, and ATP-dependent transport into bile. This efflux across the canalicular membrane is mediated by multidrug resistance protein 2 (MRP2 or ABCC2), which is a 190-kDa glycoprotein transporting with high affinity and efficiency monoglucuronosyl bilirubin and bisglucuronosyl bilirubin into bile. MRP2 is hereditarily deficient in human Dubin-Johnson syndrome. Under pathophysiological conditions such as cholestatic liver injury and MRP2 inhibition, the basolateral efflux pump multidrug resistance protein 3 (MRP3 or ABCC3) is responsible for the occurrence of conjugated hyperbilirubinemia. MRP3 is a glycoprotein with a similar molecular mass as MRP2, with 48% amino acid identity, and with overlapping substrate specificity. Human MRP3 is the only basolateral efflux pump shown to transport bilirubin glucuronides. In human and rat hepatocytes, MRP3/Mrp3 is strongly upregulated under conditions of cholestasis and MRP2 deficiency. This is in line with the concept that basolateral efflux pumps of the hepatocyte compensate for impaired canalicular efflux of compounds into bile and contribute to balance the rate of uptake or synthesis of compounds in hepatocytes with the capacity for efflux into bile.

Promoting drug discovery by collaborative innovation: a novel risk- and reward-sharing partnership between the German Cancer Research Center and Bayer HealthCare.

As a result of the increasing cost pressure on healthcare systems, the depletion of easily addressable and well-validated target groups in drug development and the requirement of public research to contribute to innovative treatment paradigms, broad partnerships between industry and academia are becoming increasingly important. However, owing to different goals and drivers, hurdles have to be overcome to exploit the full potential of such alliances. The factors that need to be taken into account during set-up and management of such alliances and the result and impact all of this has on drug discovery have not been analyzed in a systematic manner until now. This will be the focus of this review, using the strategic alliance between the German Cancer Research Center and Bayer HealthCare as an example.

Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy.

The nine multidrug resistance proteins (MRPs) represent the major part of the 12 members of the MRP/CFTR subfamily belonging to the 48 human ATP-binding cassette (ABC) transporters. Cloning, functional characterization, and cellular localization of most MRP subfamily members have identified them as ATP-dependent efflux pumps with a broad substrate specificity for the transport of endogenous and xenobiotic anionic substances localized in cellular plasma membranes. Prototypic substrates include glutathione conjugates such as leukotriene C(4) for MRP1, MRP2, and MRP4, bilirubin glucuronosides for MRP2 and MRP3, and cyclic AMP and cyclic GMP for MRP4, MRP5, and MRP8. Reduced glutathione (GSH), present in living cells at millimolar concentrations, modifies the substrate specificities of several MRPs, as exemplified by the cotransport of vincristine with GSH by MRP1, or by the cotransport of GSH with bile acids or of GSH with leukotriene B(4) by MRP4.The role of MRP subfamily members in pathophysiology may be illustrated by the MRP-mediated release of proinflammatory and immunomodulatory mediators such as leukotrienes and prostanoids. Pathophysiological consequences of many genetic variants leading to a lack of functional MRP protein in the plasma membrane are observed in the hereditary MRP2 deficiency associated with conjugated hyperbilirubinemia in Dubin-Johnson syndrome, in pseudoxanthoma elasticum due to mutations in the MRP6 (ABCC6) gene, or in the type of human earwax and osmidrosis determined by single nucleotide polymorphisms in the MRP8 (ABCC8) gene. The hepatobiliary and renal elimination of many drugs and their metabolites is mediated by MRP2 in the hepatocyte canalicular membrane and by MRP4 as well as MRP2 in the luminal membrane of kidney proximal tubules. Therefore, inhibition of these efflux pumps affects pharmacokinetics, unless compensated by other ATP-dependent efflux pumps with overlapping substrate specificities.

Channels and transporters. Mini-symposium of the Division of Medicinal Chemistry (DMC) of the Swiss Chemical Society (SCS) at the Department of Chemistry, University of Basel, May 27, 2010.

During a half-day symposium, the topic 'Channels and Transporters' was covered with five lectures, including a presentation on 'Introduction and Basics of Channels and Transporters' by Beat Ernst, lectures on structure, function and physiology of channels and transporters ('The Structural Basis for Ion Conduction and Gating in Pentameric Ligand-Gated Ion Channels' by Raimund Dutzler and 'Uptake and Efflux Transporters for Endogenous Substances and for Drugs' by Dietrich Keppler), and a case study lecture on 'Avosentan' by Werner Neidhart. The program was completed by Matthias Hediger who introduced to the audience the National Center of Competence in Research (NCCR)-TransCure in his lecture entitled 'From Transport Physiology to Identification of Therapeutic Targets'.

Vectorial transport of nucleoside analogs from the apical to the basolateral membrane in double-transfected cells expressing the human concentrative nucleoside transporter hCNT3 and the export pump ABCC4.

The identification of the transport proteins responsible for the uptake and the efflux of nucleosides and their metabolites enables the characterization of their vectorial transport and a better understanding of their absorption, distribution, and elimination. Human concentrative nucleoside transporters (hCNTs/SLC28A) are known to mediate the transport of natural nucleosides and some nucleoside analogs into cells in a sodium-dependent and unidirectional manner. On the other hand, several human multidrug resistance proteins [human ATP-binding cassette transporter, subfamily C (ABCC)] cause resistance against nucleoside analogs and mediate transport of phosphorylated nucleoside derivatives out of the cells in an ATP-dependent manner. For the integrated analysis of uptake and efflux of these compounds, we established a double-transfected Madin-Darby canine kidney (MDCK) II cell line stably expressing the human uptake transporter hCNT3 in the apical membrane and the human efflux pump ABCC4 in the basolateral membrane. The direction of transport was from the apical to the basolateral compartment, which is in line with the unidirectional transport and the localization of both recombinant proteins in the MDCKII cells. Recombinant hCNT3 mediated the transport of several known nucleoside substrates, and we identified 5-azacytidine as a new substrate for hCNT3. It is of interest that coexpression of both transporters was confirmed in pancreatic adenocarcinomas, which represent an important clinical indication for the therapeutic use of nucleoside analogs. Thus, our results establish a novel cell system for studies on the vectorial transport of nucleosides and their analogs from the apical to the basolateral compartment. The results contribute to a better understanding of the cellular transport characteristics of nucleoside drugs.

Membrane transporters in drug development.

Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.

Expression of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) is affected by genetic factors and cholestasis in human liver.

UNLABELLED: An important function of hepatocytes is the biotransformation and elimination of various drugs, many of which are organic cations and are taken up by organic cation transporters (OCTs) of the solute carrier family 22 (SLC22). Because interindividual variability of OCT expression may affect response to cationic drugs such as metformin, we systematically investigated genetic and nongenetic factors of OCT1/SLC22A1 and OCT3/SLC22A3 expression in human liver. OCT1 and OCT3 expression (messenger RNA [mRNA], protein) was analyzed in liver tissue samples from 150 Caucasian subjects. Hepatic OCTs were localized by way of immunofluorescence microscopy. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and genome-wide single-nucleotide polymorphism microarray technology served to genotype 92 variants in the SLC22A1-A3/OCT1-3 gene cluster. Transport of metformin by recombinant human OCT1 and OCT3 was compared using transfected cells. OCT1 mRNA and protein expression varied 113- and 83-fold, respectively; OCT3 mRNA expression varied 27-fold. OCT1 transcript levels were on average 15-fold higher compared with OCT3. We localized the OCT3 protein to the basolateral hepatocyte membrane and identified metformin as an OCT3 substrate. OCT1 and OCT3 expression are independent of age and sex but were significantly reduced in liver donors diagnosed as cholestatic (P < or = 0.01). Several haplotypes for OCT1 and OCT3 were identified. Multivariate analysis adjusted for multiple testing showed that only the OCT1-Arg61Cys variant (rs12208357) strongly correlated with decreased OCT1 protein expression (P < 0.0001), and four variants in OCT3 (rs2292334, rs2048327, rs1810126, rs3088442) were associated with reduced OCT3 mRNA levels (P = 0.03). CONCLUSION: We identified cholestasis and genetic variants as critical determinants for considerable interindividual variability of hepatic OCT1 and OCT3 expression. This indicates consequences for hepatic elimination of and response to OCT substrates such as metformin.

Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation.

The DNA methyltransferase inhibitors 5-azacytidine (5-azaCyd) and 5-aza-2'-deoxycytidine have found increasing use for the treatment of myeloid leukemias and solid tumors. Both nucleoside analogues must be transported into cells and phosphorylated before they can be incorporated into DNA and inactivate DNA methyltransferases. The members of the human equilibrative and concentrative nucleoside transporter families mediate transport of natural nucleosides and some nucleoside analogues into cells. However, the molecular identity of the transport proteins responsible for mediating the uptake of 5-azanucleosides has remained unknown. To this end, we have generated a stably transfected Madin-Darby canine kidney strain II cell line expressing recombinant hCNT1. An antiserum directed against hCNT1 specifically detected the protein in the apical membrane of hCNT1-expressing Madin-Darby canine kidney cells. Using [14C]5-azaCyd, we show here that hCNT1 mediated the Na+-dependent uptake of this drug with a Km value of 63 micromol/L. Na+-dependent transport of radiolabeled cytidine, uridine, and 5-fluoro-5'-deoxyuridine further showed the functionality of the transporter. hCNT1-expressing cells were significantly more sensitive to 5-azaCyd, and drug-dependent covalent trapping of DNA methyltransferase 1 was substantially more pronounced. Importantly, these results correlated with a significant sensitization of hCNT1-expressing cells toward the demethylating effects of 5-azaCyd and 5-aza-2'-deoxycytidine. In conclusion, our study identifies 5-azaCyd as a novel substrate for hCNT1 and provides direct evidence that hCNT1 is involved in the DNA-demethylating effects of this drug.

Interplay of conjugating enzymes with OATP uptake transporters and ABCC/MRP efflux pumps in the elimination of drugs.

BACKGROUND: Biliary excretion is a major elimination route of many drugs and their metabolites. Hepatobiliary elimination is a vectorial process involving uptake transporters in the basolateral hepatocyte membrane, possibly Phase I and Phase II metabolizing enzymes, and ATP-dependent efflux pumps in the apical hepatocyte membrane. OBJECTIVES: Because many drugs and their metabolites are anions, this review focuses on transporters involved in their hepatocellular uptake (members of the organic anion transporting polypeptide (OATP) family) and biliary elimination (apical conjugate efflux pump ABCC2/MRP2). METHODS: The molecular and functional characteristics of the human OATP and ABCC/MRP transporters are presented, including a detailed overview of endogenous and drug substrates. Examples illustrate the interplay of transporters with Phase II conjugating enzymes. Model systems to study the vectorial transport of organic anions are also discussed. RESULTS/CONCLUSIONS: OATP uptake transporters, conjugating enzymes, and ABCC2/MRP2 work in concert to enable the hepatobiliary elimination of anionic drugs and their metabolites. It is increasingly important to understand how genetic variants of these transporters and enzymes influence the interindividual variability of drug elimination.

Vectorial transport of the plant alkaloid berberine by double-transfected cells expressing the human organic cation transporter 1 (OCT1, SLC22A1) and the efflux pump MDR1 P-glycoprotein (ABCB1).

An important function of hepatocytes is the biliary elimination of endogenous and xenobiotic small molecules, many of which are organic cations. To study this vectorial transport of organic cations, we constructed a double-transfected Madin-Darby canine kidney strain II (MDCKII) cell line permanently expressing the human organic cation transporter 1 (OCT1, SLC22A1) in the basolateral membrane and MDR1 P-glycoprotein (MDR1 P-gp, ABCB1), an adenosine triphosphate (ATP)-dependent efflux pump for organic cations, in the apical membrane. Additionally, MDCKII single transfectants stably expressing OCT1, MDR1 P-gp, or human organic cation transporter 2 (OCT2, SLC22A2) were generated. Antisera directed against OCT1 or OCT2 specifically detected OCT1 in the basolateral membrane of human hepatocytes, OCT2 in tubular epithelial cells of human kidney, and the respective recombinant transporter in the basolateral membrane of MDCKII transfectants. We identified the lipophilic organic cation berberine, a fluorescent plant alkaloid exhibiting a broad range of biological activities, as substrate of OCT1 and OCT2 with Michaelis-Menten constants of 14.8 microM and 4.4 microM, respectively. Berberine also inhibited the uptake of the prototypic cations tetraethylammonium and 1-methyl-4-phenylpyridinium by MDCK-OCT1 and MDCK-OCT2 transfectants. When transfected cells were grown polarized on permeable filter supports, berberine was transferred from the basolateral to the apical compartments many times faster by MDCK-OCT1/MDR1 P-gp double transfectants than by MDCK-OCT1 or MDCK-MDR1 P-gp single transfectants. The specific MDR1 P-gp inhibitor, zosuquidar trihydrochloride (LY335979), strongly inhibited berberine efflux into the apical compartment. The MDCK-OCT1/MDR1 P-gp double transfectants may be useful to identify additional cationic substrates and inhibitors of OCT1 and MDR1 P-gp, including drug candidates.

ATP-dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4).

The proinflammatory mediators leukotriene (LT) B(4) and LTC(4) must be transported out of cells before they can interact with LT receptors. Previously, we identified the multidrug resistance protein ABCC1 (MRP1) as an efflux pump for LTC(4). However, the molecular basis for the efflux of LTB(4) was unknown. Here, we demonstrate that human ABCC4 mediates the ATP-dependent efflux of LTB(4) in the presence of reduced glutathione (GSH), whereby the latter can be replaced by S-methyl GSH. Transport studies were performed with inside-out membrane vesicles from V79 fibroblasts and Sf9 insect cells that contained recombinant ABCC4, with vesicles from human platelets and myelomonocytic U937 cells, which were rich in endogenous ABCC4, but ABCC1 was below detectability. Moreover, human polymorphonuclear leukocytes contained ABCC4. K(m) values for LTB(4) were 5.2 muM with vesicles from fibroblasts and 5.6 muM with vesicles from platelets. ABCC4, with its broad substrate specificity, also functioned as an ATP-dependent efflux pump for LTC(4) with a K(m) of 0.13 muM in vesicles from fibroblasts and 0.32 muM in vesicles from platelets. However, GSH was not required for the transport of this glutathionylated leukotriene. The transport of LTC(4) by ABCC4 explains its release from platelets during transcellular synthesis. ATP-dependent transport of LTB(4) and LTC(4) by ABCC4 was inhibited by several organic anions, including S-decyl GSH, sulindac sulfide, and by the LTD(4) receptor antagonists montelukast and 3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-((3-dimethyl-amino-3-oxopropyl)-thio)-methyl)thio)propanoic acid (MK571). Thus, as an efflux pump for the proinflammatory mediators LTB(4) and LTC(4), ABCC4 may represent a novel target for anti-inflammatory therapies.

Data-based mathematical modeling of vectorial transport across double-transfected polarized cells.

Vectorial transport of endogenous small molecules, toxins, and drugs across polarized epithelial cells contributes to their half-life in the organism and to detoxification. To study vectorial transport in a quantitative manner, an in vitro model was used that includes polarized MDCKII cells stably expressing the recombinant human uptake transporter OATP1B3 in their basolateral membrane and the recombinant ATP-driven efflux pump ABCC2 in their apical membrane. These double-transfected cells enabled mathematical modeling of the vectorial transport of the anionic prototype substance bromosulfophthalein (BSP) that has frequently been used to examine hepatobiliary transport. Time-dependent analyses of (3)H-labeled BSP in the basolateral, intracellular, and apical compartments of cells cultured on filter membranes and efflux experiments in cells preloaded with BSP were performed. A mathematical model was fitted to the experimental data. Data-based modeling was optimized by including endogenous transport processes in addition to the recombinant transport proteins. The predominant contributions to the overall vectorial transport of BSP were mediated by OATP1B3 (44%) and ABCC2 (28%). Model comparison predicted a previously unrecognized endogenous basolateral efflux process as a negative contribution to total vectorial transport, amounting to 19%, which is in line with the detection of the basolateral efflux pump Abcc4 in MDCKII cells. Rate-determining steps in the vectorial transport were identified by calculating control coefficients. Data-based mathematical modeling of vectorial transport of BSP as a model substance resulted in a quantitative description of this process and its components. The same systems biology approach may be applied to other cellular systems and to different substances.

Involvement of mitogen-activated protein kinase signaling pathways in microcystin-LR-induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3.

The serine/threonine protein phosphatase (PP) 2A inhibitor, microcystin-LR, selectively induces liver damage and promotes hepatocarcinogenesis. It is thought that microcystin-LR affects hepatocellular viability mainly through inhibition of PP2A, partially through PP1, and, in addition, by generation of reactive oxygen species (ROS). However, the molecular basis of the selective liver damage and the balance between cell death and survival remained unclear. We analyzed the cytotoxicity of low doses of microcystin-LR using HEK293 cells stably expressing the human hepatocyte uptake transporters, organic anion transporting polypeptide (OATP)1B1 (HEK293-OATP1B1 cells) and OATP1B3 (HEK293-OATP1B3 cells). HEK293-OATP1B1 (IC(50) 6.6nM) and HEK293-OATP1B3 cells (IC(50) 6.5nM) were equally very sensitive to microcystin-LR. In contrast, control-vector-transfected (HEK293-CV) cells were resistant to microcystin-LR. Using HEK293-OATP1B3 cells, the cytotoxicity was attenuated by substrates and inhibitors of OATP1B3, including bromosulfophthalein, rifampicin, and cyclosporin A. Microcystin-LR was transported into HEK293-OATP1B3 cells with 1.2 microM Km value, and its uptake was inhibited by above substances. Accumulation of microcystin-LR in the HEK293-OATP1B1 and HEK293-OATP1B3 cells was increased in a dose-dependent manner but not in HEK293-CV cells. Cellular serine/threonine PP activity of HEK293-OATP1B3 cells was decreased by microcystin-LR but not in HEK293-CV cells. Apoptotic changes were observed after incubation of the HEK293-OATP1B3 cells with microcystin-LR. We found by FACS analysis that microcystin-LR induced apoptosis but not necrosis in HEK293-OATP1B3 cells. Microcystin-LR activated several mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK, and p38 through inhibition of PP2A. In addition, the cytotoxicity of microcystin-LR was attenuated by the inhibitors of MAPK pathways, including U0126, SP600125, and SB203580. The ROS scavenger N-acetyl-L-cysteine partially attenuated the cytotoxicity of microcystin-LR. Thus, the present study demonstrates that microcystin-LR induces apoptosis through activation of multiple MAPK pathways subsequent to its selective uptake via OATP1B1 and OATP1B3 and followed by inhibition of PP2A, in addition to the ROS generation which might contribute to apoptosis.

The apical conjugate efflux pump ABCC2 (MRP2).

ABCC2 is a member of the multidrug resistance protein subfamily localized exclusively to the apical membrane domain of polarized cells, such as hepatocytes, renal proximal tubule epithelia, and intestinal epithelia. This localization supports the function of ABCC2 in the terminal excretion and detoxification of endogenous and xenobiotic organic anions, particularly in the unidirectional efflux of substances conjugated with glutathione, glucuronate, or sulfate, as exemplified by leukotriene C(4), bilirubin glucuronosides, and some steroid sulfates. The hepatic ABCC2 pump contributes to the driving forces of bile flow. Acquired or hereditary deficiency of ABCC2, the latter known as Dubin-Johnson syndrome in humans, causes an increased concentration of bilirubin glucuronosides in blood because of their efflux from hepatocytes via the basolateral ABCC3, which compensates for the deficiency in ABCC2-mediated apical efflux. In this article we provide an overview on the molecular characteristics of ABCC2 and its expression in various tissues and species. We discuss the transcriptional and posttranscriptional regulation of ABCC2 and review approaches to the functional analysis providing information on its substrate specificity. A comprehensive list of sequence variants in the human ABCC2 gene summarizes predicted and proven functional consequences, including variants leading to Dubin-Johnson syndrome.

Vectorial transport of enalapril by Oatp1a1/Mrp2 and OATP1B1 and OATP1B3/MRP2 in rat and human livers.

Although Oatp1a1 (rat organic anion-transporting polypeptide 1a1) was the transporter found responsible for the hepatocellular entry of enalapril (EN) into the rat liver, the canalicular transporter involved for excretion of EN and the metabolite, enalaprilat (ENA), was unknown. The Eisai hyperbilirubinemic rat (EHBR) that lacks Mrp2 (multidrug resistance-associated protein 2) was used to appraise the role of Mrp2 in the excretion of [3H]EN and its metabolite [3H]ENA in single-pass rat liver preparations. Although the total and metabolic clearances and hepatic extraction ratios at steady-state were virtually unaltered for EN in EHBR compared with published values of Sprague-Dawley rats, the biliary clearances of EN and ENA were significantly reduced almost to zero (P<0.05). Involvement of human OATP1B1, OATP1B3, and MRP2 in EN transport was further assessed in single- or double-transfected mammalian cells. Human embryonic kidney 293 cells that expressed OATP1B1 or OATP1B3 showed that OATP1B3 transport of EN (20-500 microM) was of low affinity, whereas transport of EN by OATP1B1 was associated with the Km of 262+/-35 microM, a value similar to that for Oatp1a1 (214 microM). The transcellular transport of EN via human OATP1B1 and MRP2, investigated with the double-transfected Madin-Darby canine kidney (MDCK) II cells in the Transwell system, showed that the sinusoidal to canalicular flux of EN in the OATP1B1/MRP2/MDCK cells was significantly higher (P<0.05) than that of mock/MDCK and OATP1B1/MDCK cells. EN was transported by Oatp1a1 and Mrp2 in rats and OATP1B1/OATP1B3 and MRP2 in humans.

Molecular characterization and inhibition of amanitin uptake into human hepatocytes.

Amatoxins are the main poison of the green death cap (Amanita phalloides) and among the most dangerous natural toxins causing hepatic failure. A possible therapeutic approach is the inhibition of the transporting systems mediating the uptake of amatoxins into human hepatocytes, which, however, have yet to be identified. In the current study we tested whether members of the organic anion-transporting polypeptide (OATP) family, localized in the sinusoidal membranes of human hepatocytes, are involved in amatoxin uptake. For this, Madin Darby canine kidney strain II (MDCKII) cells stably expressing human OATP1B3, OATP2B1, or OATP1B1, were assayed for the uptake of 3H-labeled O-methyl-dehydroxymethyl-alpha-amanitin. Under our conditions, only OATP1B3 was able to transport amanitin with a K(m) value of 3.7 microM +/- 0.6 microM. Accordingly, toxin uptake was inhibited by OATP1B3 substrates and inhibitors (cyclosporin A, rifampicin, the quinoline derivatives MK571 ([(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid]) and montelukast, the cholecystokinin octapeptide (CCK-8), paclitaxel, and bromosulfophthalein), as well as by some antidotes used in the past for the treatment of human amatoxin poisoning (silibinin dihemisuccinate, penicillin G, prednisolone phosphate, and antamanide). These transport studies are in line with viability assays monitoring the toxic effect of amanitin on the transfected MDCKII cells. Further support for amatoxin transport was found in primary human hepatocytes, expressing OATP1B3, OATP2B1, and OATP1B1, where CCK-8, a substrate specific for OATP1B3, prevented the fragmentation of nucleoli, a lesion typical for amanitin action. In conclusion, we have identified OATP1B3 as the human hepatic uptake transporter for amatoxins; moreover, substrates and inhibitors of OATP1B3, among others rifampicin, may be useful for the treatment of human amatoxin poisoning.

Substrate specificity of human ABCC4 (MRP4)-mediated cotransport of bile acids and reduced glutathione.

The multidrug resistance protein ABCC4 (MRP4), a member of the ATP-binding cassette superfamily, mediates ATP-dependent unidirectional efflux of organic anions out of cells. Previous studies showed that human ABCC4 is localized to the sinusoidal membrane of hepatocytes and mediates, among other substrates, the cotransport of reduced glutathione (GSH) with bile acids. In the present study, using inside-out membrane vesicles, we demonstrated that human ABCC4 in the presence of physiological concentrations of GSH has a high affinity for the taurine and glycine conjugates of the common natural bile acids as well as the unconjugated bile acid cholate. Chenodeoxycholyltaurine and chenodeoxycholylglycine were the GSH cosubstrates with the highest affinities for ABCC4, with K(m) values of 3.6 and 5.9 microM, respectively. Ursodeoxycholyltaurine and ursodeoxycholylglycine were cotransported together with GSH by ABCC4 with K(m) values of 7.8 and 12.5 microM, respectively, but no transport of ursodeoxycholate and deoxycholate was observed. The simultaneous transport of labeled GSH and cholyltaurine or cholylglycine was demonstrated in double-labeled cotransport experiments with a bile acid-to-GSH ratio of approximately 1:22. K(m) values of the bile acids for ABCC4 were in a range similar to those reported for the canalicular bile salt export pump ABCB11. Under physiological conditions, the sinusoidal ABCC4 may compete with canalicular ABCB11 for bile acids and thereby play a key role in determining the hepatocyte concentration of bile acids. In cholestatic conditions, ABCC4 may become a key pathway for efflux of bile acids from hepatocytes into blood.

ABCC drug efflux pumps and organic anion uptake transporters in human gliomas and the blood-tumor barrier.

Delivery of therapeutic agents to the brain and its neoplasms depends on the presence of membrane transport proteins in the blood-brain barrier and in the target cells. The cellular and subcellular localization of these membrane transporters determines the drug accessibility to the brain and its tumors. We therefore analyzed the expression and localization of six members of the multidrug resistance protein family of ATP-dependent efflux pumps (ABCC1-ABCC6, formerly MRP1-MRP6) and of six organic anion uptake transporters (OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2B1, and OATP4A1) in 61 human glioma specimens of different histologic subtypes. Real-time PCRs indicated expressions of ABCC1, ABCC3, ABCC4, and ABCC5. In addition, we detected expressions of the OATP uptake transporter genes SLCO1A2, SLCO1C1, SLCO2B1, and SLCO4A1. At the protein level, however, only OATP1A2 and OATP2B1 were detectable by immunofluorescence microscopy in the luminal membrane of endothelial cells forming the blood-brain barrier and the blood-tumor barrier, but not in the glioma cells. ABCC4 and ABCC5 proteins were the major ABCC subfamily members in gliomas, localized both at the luminal side of the endothelial cells and in the glioma cells of astrocytic tumors and in the astrocytic portions of oligoastrocytomas. These results indicate that expression of ABCC4 and ABCC5 is associated with an astrocytic phenotype, in accordance with their expression in astrocytes and with the higher chemoresistance of astrocytic tumors as compared with oligodendrogliomas. Our data provide a basis for the assessment of the role of uptake transporters and efflux pumps in the accessibility of human gliomas for chemotherapeutic agents.