RESUMO
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.
Assuntos
Ductos Biliares/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Colestase/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Proteína 2 Associada à Farmacorresistência MúltiplaRESUMO
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.
Assuntos
Hiperbilirrubinemia Hereditária/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Animais , Bile/metabolismo , Bilirrubina/análogos & derivados , Bilirrubina/sangue , Transporte Biológico , Hepatócitos/metabolismo , Humanos , Hiperbilirrubinemia Hereditária/sangue , Icterícia Idiopática Crônica/sangue , Icterícia Idiopática Crônica/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Proteína 2 Associada à Farmacorresistência Múltipla , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
The ezrin-radixin-moesin (ERM) family of proteins crosslink actin filaments and integral membrane proteins. Radixin (encoded by Rdx) is the dominant ERM protein in the liver of wildtype mice and is concentrated at bile canalicular membranes (BCMs). Here we show that Rdx(-/-) mice are normal at birth, but their serum concentrations of conjugated bilirubin begin to increase gradually around 4 weeks, and they show mild liver injury after 8 weeks. This phenotype is similar to human conjugated hyperbilirubinemia in Dubin-Johnson syndrome, which is caused by mutations in the multidrug resistance protein 2 (MRP2, gene symbol ABCC2), although this syndrome is not associated with overt liver injury. In wildtype mice, Mrp2 concentrates at BCMs to secrete conjugated bilirubin into bile. In the BCMs of Rdx(-/-) mice, Mrp2 is decreased compared with other BCM proteins such as dipeptidyl peptidase IV (CD26) and P-glycoproteins. In vitro binding studies show that radixin associates directly with the carboxy-terminal cytoplasmic domain of human MRP2. These findings indicate that radixin is required for secretion of conjugated bilirubin through its support of Mrp2 localization at BCMs.
Assuntos
Proteínas Sanguíneas/fisiologia , Proteínas do Citoesqueleto/fisiologia , Hiperbilirrubinemia/genética , Proteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Canalículos Biliares/citologia , Canalículos Biliares/metabolismo , Canalículos Biliares/ultraestrutura , Proteínas Sanguíneas/deficiência , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Proteínas do Citoesqueleto/deficiência , Resistência a Múltiplos Medicamentos/genética , Glutationa Transferase/metabolismo , Hiperbilirrubinemia/patologia , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Microvilosidades/ultraestrutura , Proteína 2 Associada à Farmacorresistência Múltipla , Mutação , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Preparações Farmacêuticas/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico , Predisposição Genética para Doença , Variação Genética , Humanos , Rim/metabolismo , Rim/fisiopatologia , Fígado/metabolismo , Fígado/fisiopatologia , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , FenótipoRESUMO
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.
Assuntos
Colestase/metabolismo , Fígado/metabolismo , Fator 1 de Transcrição de Octâmero/genética , Fator 1 de Transcrição de Octâmero/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transporte Biológico/fisiologia , Colestase/patologia , Frequência do Gene/genética , Glucose/metabolismo , Haplótipos/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Hipoglicemiantes/farmacocinética , Fígado/patologia , Metformina/farmacocinética , Análise Multivariada , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/metabolismo , População Branca/genéticaRESUMO
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.
Assuntos
Transporte Biológico/genética , Proteínas de Membrana Transportadoras/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Nucleosídeos/metabolismo , Animais , Azacitidina/metabolismo , Linhagem Celular Transformada , Cães , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
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'.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte/fisiologia , Canais Iônicos/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Congressos como Assunto , HumanosRESUMO
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.
Assuntos
Azacitidina/metabolismo , Metilação de DNA , DNA/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Azacitidina/análogos & derivados , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Cães , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , Leucotrieno B4/metabolismo , Leucotrieno C4/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Plaquetas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Eritrócitos/metabolismo , Glutationa/farmacologia , Humanos , Leucócitos Mononucleares/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neutrófilos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Berberina/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico , Linhagem Celular , Dibenzocicloeptenos/farmacologia , Cães , Imunofluorescência , Humanos , Rim/metabolismo , Fígado/metabolismo , Especificidade de Órgãos , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico , Quinolinas/farmacologia , TransfecçãoRESUMO
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.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Transporte Biológico Ativo , Linhagem Celular , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Ânions Orgânicos/genética , Especificidade por Substrato , TransfecçãoRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Microcistinas/metabolismo , Microcistinas/toxicidade , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Transportadores de Ânions Orgânicos Sódio-Independentes/fisiologia , Transportadores de Ânions Orgânicos/fisiologia , Transdução de Sinais/fisiologia , Acetilcisteína/farmacologia , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Citometria de Fluxo , Humanos , Indicadores e Reagentes , Transportador 1 de Ânion Orgânico Específico do Fígado , Toxinas Marinhas , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica , Neoplasias Encefálicas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Oligodendroglioma/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Adulto , Idoso , Astrocitoma/tratamento farmacológico , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Humanos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Fluorescência , Pessoa de Meia-Idade , Oligodendroglioma/tratamento farmacológico , Oligodendroglioma/patologia , Frações SubcelularesRESUMO
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.
Assuntos
Amanitinas/metabolismo , Hepatócitos/metabolismo , Animais , Bovinos , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Hepatócitos/ultraestrutura , Humanos , Transportadores de Ânions Orgânicos/metabolismo , Propionatos/farmacologia , Quinolinas/farmacologia , Sincalida/farmacologiaRESUMO
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. The cloning and stable expression of the complementary DNAs encoding the organic anion transporters in the basolateral membrane of human hepatocytes and of members of the ABCC subfamily of efflux pumps in the apical as well as in the basolateral membrane have improved our understanding of hepatobiliary elimination and of the substrate specificity with respect to anionic conjugates. The stable expression of human hepatocyte uptake and efflux transporters in polarized cell lines, as described in this chapter, provides valuable tools for the in vitro analysis of human hepatobiliary transport in general and specifically for uptake and efflux of the anionic conjugates formed in various phase 2 reactions.
Assuntos
Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Membrana Celular/química , Cães , Humanos , Inativação Metabólica , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/classificação , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/genética , Xenobióticos/metabolismo , Xenobióticos/farmacologiaRESUMO
OBJECTIVE: Hepatocellular uptake transporters are involved in the hepatobiliary elimination of endogenous and xenobiotic substances. Mutations in genes encoding these uptake transporters may be key determinants of interindividual variability in hepatobiliary elimination and drug disposition. Our aim was to investigate the functional consequences of mutations in the SLCO1B3 gene encoding the hepatic uptake transporter for organic anions OATP1B3, formerly termed OATP8. METHODS: Mutations occurring in Caucasian Europeans and observed in databases were introduced into the SLCO1B3 cDNA and the consequences were analyzed in stably transfected canine MDCKII cells and human HEK293 cells. The functional consequences were examined for two frequent polymorphisms SLCO1B3-334T>G, encoding OATP1B3-S112A (allelic frequency of 74%) and SLCO1B3-699G>A, encoding OATP1B3-M233I (allelic frequency of 71%) and one rare polymorphism SLCO1B3-1564G>T, encoding OATP1B3-G522C (allelic frequency of 1.9%) and one artificial mutation SLCO1B3-1748G>A, encoding OATP1B3-G583E. RESULTS: OATP1B3-S112A, OATP1B3-M233I, and the OATP1B3 protein corresponding to the reference sequence (accession NM_019844), showed a comparable lateral localization in stably transfected MDCKII cells, whereas OATP1B3-G522C and OATP1B3-G583E proteins were retained intracellularly. Both latter amino acid substitutions abolished the transport of bile acids mediated by OATP1B3, whereas other substrates, like bromosulfophthalein, were transported by all polymorphic variants of the protein. CONCLUSIONS: The functional consequences of three polymorphisms and one artificial mutation include differences in the localization and in transport characteristics of several OATP1B3 proteins. This study demonstrates the importance of the analysis of genetic variations in genes encoding transport proteins for the understanding of individual variations in the hepatobiliary elimination of substances.
Assuntos
Hepatócitos/metabolismo , Mutação/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Polimorfismo Genético/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico/genética , Linhagem Celular , Primers do DNA , Cães , Europa (Continente) , Frequência do Gene , Humanos , Fígado/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Transportadores de Ânions Orgânicos Sódio-Independentes/química , Conformação Proteica , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Transfecção , População Branca/genéticaRESUMO
The human multidrug resistance protein 3 (MRP3, symbol ABCC3) is an ATP-binding cassette transporter that mediates the efflux of organic anions, including lipophilic substances conjugated with glucuronate, sulphate or glutathione, across the basolateral membrane of polarized cells (e.g. hepatocytes) into blood. Genetic variants of MRP3 may affect the transport of these substances out of cells. The aims of this study were: (i) to identify MRP3 polymorphisms; (ii) to functionally characterize one relatively frequent MRP3 polymorphism; and (iii) to establish whether MRP3 transports bilirubin glucuronosides. Exonic nucleotide variants in the ABCC3 gene were identified by single-strand conformation polymorphism analysis. The 3890G>A mutation, resulting in MRP3-ArgHis, was introduced into the ABCC3 cDNA which was stably transfected into MDCKII cells. For the functional characterization of MRP3-ArgHis in comparison with MRP3, ATP-dependent transport was analysed in isolated membrane vesicles. Two non-synonymous MRP3 variants were identified with an allele frequency of 0.003 for 1643T>A (MRP3-LeuGln) and 0.08 for 3890G>A (MRP3-ArgHis). Because of the high frequency of the 3890G>A mutation, and because of the close proximity of Arg to the second nucleotide-binding domain, we pursued the functional characterization of the MRP3-ArgHis polymorphic variant. MRP3-ArgHis was correctly localized to the basolateral membrane of polarized MDCKII cells. We identified monoglucuronosyl bilirubin, bisglucuronosyl bilirubin and leukotriene C4 as substrates for both MRP3 and MRP3-ArgHis. Dehydroepiandrosterone-3-sulphate and 17beta-glucuronosyl oestradiol were transported with similar kinetics by MRP3 and MRP3-ArgHis. This experimental setup provides a useful tool to analyse the functional consequences of polymorphic variants of MRP3.
Assuntos
Arginina/química , Histidina/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Células Cultivadas , Primers do DNA , Cães , Imunofluorescência , Humanos , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutagênese Sítio-Dirigida , Polimorfismo Conformacional de Fita SimplesRESUMO
Hyperbilirubinemia is a frequent side effect induced by long-term therapy with the antibiotic fusidate. The aim of this study was to elucidate the molecular mechanisms of fusidate-induced hyperbilirubinemia by investigating its influence on hepatic transport systems in the canalicular membrane. Using canalicular membrane vesicles from rat liver, we determined the effect of fusidate on the adenosine 5'-triphosphate (ATP)-dependent transport of substrates of the apical conjugate export pump, multi-drug resistance protein 2 (Mrp2, symbol Abcc2) and the bile salt export pump (Bsep, symbol Abcb11). Fusidate inhibited the ATP-dependent transport of the Mrp2 substrates 17beta-glucuronosyl estradiol and leukotriene C4, and the transport of cholyltaurine by Bsep with Ki values of 2.2+/-0.3, 7.6+/-1.3, and 5.5+/-0.8 microM, respectively. To elucidate the in vivo implication of these findings, the effect of fusidate treatment on the elimination of intravenously administered tracer doses of 17beta-glucuronosyl estradiol and cholyltaurine into bile was studied in rats. Treatment with fusidate (100 micromol/kg body weight) reduced the biliary excretion rate of 17beta-glucuronosyl [3H]estradiol and [3H]cholyltaurine by 75 and 80%, respectively. Extended treatment of rats with fusidate (100 micromol/kg body weight, three times daily i.p. for 3 days) reduced hepatic Mrp2 protein levels by 61% (P<0.001). Our data suggest that there are at least two different mechanisms involved in the impairment of transport processes and hepatobiliary elimination by fusidate, direct inhibition of transport of Mrp2 and Bsep substrates by competitive interaction and impairment by a decreased level of hepatic Mrp2.
Assuntos
Antibacterianos/farmacologia , Estradiol/análogos & derivados , Ácido Fusídico/farmacologia , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Proteínas de Membrana Transportadoras , Trifosfato de Adenosina/metabolismo , Animais , Canalículos Biliares/citologia , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Interações Medicamentosas , Estradiol/farmacologia , Glucuronatos/farmacologia , Hepatócitos/metabolismo , Fígado/metabolismo , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Coelhos , Ratos , Ratos Wistar , Ácido Taurocólico/farmacologia , Transfecção , TrítioRESUMO
BACKGROUND: Cysteinyl leukotrienes (LTs) are potent proinflammatory mediators. They are predominantly excreted from blood by hepatobiliary elimination. To explore the clinical significance of biliary cysteinyl LTs, we determined their concentration changes in bile during treatment in patients with obstructive jaundice. METHODS: Bile samples were obtained during endoscopic or transhepatic biliary drainage. Leukotrienes C(4), D(4), and E(4) were quantified by two-step reversed-phase high-performance liquid chromatography and subsequent radioimmunoassay. RESULTS: The increased excretion of cysteinyl LTs (LTC(4) + LTD(4) + LTE(4)) decreased between day 1 and 14 after drainage (means, 171 pmol/h to 79 pmol/h; P < 0.02). During drainage, the excretion was higher when there was additional cholangitis (mean, 225 and 86 pmol/h, with and without cholangitis, respectively; P < 0.001). The concentrations of LTD(4) and LTE(4) were also higher with additional cholangitis than without (LTD(4), mean 6.0 vs 2.0 nM; P < 0.05; LTE(4), 6.8 vs 2.4 nM; P < 0.02, respectively). Biliary LTC(4) was detected only in patients with cholangitis. The biliary excretion of cysteinyl LTs was positively correlated with leukocyte concentration ( r = 0.68; P < 0.005) and C-reactive protein ( r = 0.73; P < 0.005) in blood. Furthermore, only in the absence of cholangitis, the excretion was positively correlated with serum gamma-glutamyl transferase ( r = 0.76; P < 0.02) and alanine aminotransferase ( r = 0.72; P < 0.02). CONCLUSIONS: The excretion of biliary cysteinyl LTs increases with the severity of cholestasis and hepatic inflammation in patients with obstructive jaundice. An additional increase of cysteinyl LTs was observed during bacterial cholangitis. The increased biliary excretion of biologically active cysteinyl LTs may contribute to the aggravation of cholestasis and inflammatory reaction in obstructive jaundice.