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.

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.

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.

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'.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

Uptake and efflux transporters for conjugates in human hepatocytes.

Conjugates of endogenous substances and of xenobiotics, formed extrahepatically or inside hepatocytes, undergo vectorial transport into bile. Substances conjugated with glucuronate, sulfate, or glutathione are substrates for organic anion uptake transporters in the basolateral (sinusoidal) membrane as well as substrates for the unidirectional ATP-driven conjugate efflux pump in the apical (canalicular) membrane, termed multidrug resistance protein 2 (MRP2; systematic name ABCC2). Localization of the efflux pumps ABCC3 and ABCC4 to the basolateral membrane of human hepatocytes has provided insight into the molecular mechanisms of conjugate efflux from hepatocytes into blood, as exemplified by the efflux of bilirubin glucuronosides mediated by ABCC3.

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.

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.

Prostanoid transport by multidrug resistance protein 4 (MRP4/ABCC4) localized in tissues of the human urogenital tract.

Purpose: The seminal vesicles are the major source of prostaglandins in seminal fluid. For prostanoid action on cell surfaces they must be released from synthesizing cells. MRP4/ABCC4 (multidrug resistance protein 4 adenosine triphosphate-binding cassette, subfamily C, member 4) is an adenosine triphosphate dependent export pump for organic anions that may mediate prostanoid transport across the plasma membranes.

Human hepatobiliary transport of organic anions analyzed by quadruple-transfected cells.

Hepatobiliary elimination of many organic anions is initiated by OATP1B1 (OATP2, LST-1, OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), which are the predominant uptake transporters of human hepatocytes. Thereafter, the unidirectional efflux pump ABCC2 (multidrug resistance protein 2) mediates the transport of organic anions, including glutathione conjugates and glucuronosides, into bile. In this study, we generated a Madin-Darby canine kidney (MDCKII) cell line stably expressing recombinant OATP1B1, OATP1B3, and OATP2B1 in the basolateral membrane and ABCC2 in the apical membrane.

Expression and localization of hepatobiliary transport proteins in progressive familial intrahepatic cholestasis.

Mutations of the bile salt export pump (BSEP) or the multidrug resistance P-glycoprotein 3 (MDR3) are linked to impaired bile salt homeostasis and lead to progressive familial intrahepatic cholestasis (PFIC)-2 and -3, respectively. The regulation of bile salt transporters in PFIC is not known. Expression of hepatobiliary transporters in livers of ten patients with a PFIC phenotype was studied by quantitative reverse transcription polymerase chain reaction, Western blotting, and immunofluorescence microscopy.

Expression and localization of human multidrug resistance protein (ABCC) family members in pancreatic carcinoma.

Pancreatic ductal adenocarcinoma is among the top 10 causes of death from cancer in industrialized countries. In comparison with other gastrointestinal malignancies, pancreatic cancer is one of the tumors most resistant to chemotherapy. An important mechanism of tumor multidrug resistance is increased drug efflux mediated by several transporters of the ABC superfamily.