estrone-sulfate and estradiol-17-beta-glucuronide

Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda -derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), estradiol-17β-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a K

Topics: Amino Acid Sequence; Animals; ATP-Binding Cassette Transporters; Cholesterol; Estradiol; Estrone; Fluoresceins; Phylogeny; Quinidine; Sequence Alignment; Sf9 Cells; Spodoptera

The U.S. President Barack Obama has announced, in his State of the Union address on January 20, 2015, the Precision Medicine Initiative, a US$215-million program. For global precision medicine to become a reality, however, biological and environmental "variome" in previously understudied populations ought to be mapped and catalogued. Chief among the molecular targets that warrant global mapping is the organic anion-transporting polypeptide 1B1 (OATP1B1), encoded by solute carrier organic anion transporter family member 1B1 (SLCO1B1), a hepatic uptake transporter predominantly expressed in the basolateral side of hepatocytes. Human OATP1B1 plays a crucial role in the transport of a wide variety of substrates. This includes endogenous compounds such as bile salts as well as medicines, including benzylpenicillin, methotrexate, pravastatin, and rifampicin, and natural toxins microcystin and phalloidin. Genetic variations observed in the SLCO1B1 gene have been associated with altered in vitro and in vivo OATP1B1 transport activity, and consequently influencing patients' response to medicines, toxins, and susceptibility to common complex diseases. Well-characterized haplotypes, *5 (RS4149056C) and *15 (RS4149056T), have been associated with a strikingly reduced uptake of multiple OATP1B1 substrates, including estrone-3-sulfate, estradiol-17β-d-glucuronide, atorvastatin, cerivastatin, pravastatin, and rifampicin. In particular, RS4149056C is observed in 60% of the Cape admixed (CA) population and is associated with increased plasma concentrations of many statins as well as fexofenadine and repaglinide. We designed and optimized a SNaPshot minisequencing panel to characterize the variants of relevance for precision medicine in the clinic. We report here the first study on allele and genotype frequencies for 10 nonsynonymous, 4 synonymous, and 6 intronic single-nucleotide polymorphisms of SLCO1B1 in the Zulu and CA populations of South Africa. These variants are further contextualized here, in relation to their potential clinical relevance. These observations collectively contribute to current efforts to advance global precision medicine in understudied populations and resource-limited regions of the world.

Topics: Alleles; Chromosome Mapping; Estradiol; Estrone; Genetic Variation; Genotype; Haplotypes; Humans; Liver-Specific Organic Anion Transporter 1; Polymorphism, Single Nucleotide; Precision Medicine; South Africa

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of many drugs, and the evaluation of OATP1B1-mediated drug-drug interactions (DDIs) is emphasized in the latest DDI (draft) guidance documents from U.S. and E.U. regulatory agencies. It has been suggested that some OATP1B1 inhibitors show a discrepancy in their inhibitory potential, depending on the substrates used in the cell-based assay. In this study, inhibitory effects of 14 compounds on the OATP1B1-mediated uptake of the prototypical substrates [³H]estradiol-17β-glucuronide (E₂G), [³H]estrone-3-sulfate (E₁S), and [³H]sulfobromophthalein (BSP) were studied in OATP1B1-transfected cells. Inhibitory potencies of tested compounds varied depending on the substrates. Ritonavir, gemfibrozil, and erythromycin caused remarkable substrate-dependent inhibition with up to 117-, 14-, and 13-fold difference in their IC₅₀ values, respectively. Also, the clinically relevant OATP inhibitors rifampin and cyclosporin A exhibited up to 12- and 6-fold variation in their IC₅₀ values, respectively. Regardless of the inhibitors tested, the most potent OATP1B1 inhibition was observed when [³H]E₂G was used as a substrate. Mutual inhibition studies of OATP1B1 indicated that E₂G and E₁S competitively inhibited each other, whereas BSP noncompetitively inhibited E₂G uptake. In addition, BSP inhibited E₁S in a competitive manner, but E₁S caused an atypical kinetics on BSP uptake. This study showed substrate-dependent inhibition of OATP1B1 and demonstrated that E₂G was the most sensitive in vitro OATP1B1 probe substrate among three substrates tested. This will give us an insight into the assessment of clinically relevant OATP1B1-mediated DDI in vitro with minimum potential of false-negative prediction.

Topics: Biological Transport; Cell Line; Estradiol; Estrone; HEK293 Cells; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kinetics; Liver-Specific Organic Anion Transporter 1; Organic Anion Transporters; Sulfobromophthalein

The hepatic organic anion transporting polypeptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug-drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1. The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors.

Topics: Atorvastatin; Biological Transport; Drug Interactions; Estradiol; Estrone; HEK293 Cells; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; In Vitro Techniques; Least-Squares Analysis; Liver; Liver-Specific Organic Anion Transporter 1; Models, Molecular; Multivariate Analysis; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Protein Isoforms; Pyrroles; Solute Carrier Organic Anion Transporter Family Member 1B3; Structure-Activity Relationship; Transfection

MRP2 is an efflux transporter that is expressed mainly in the canalicular membrane of hepatocytes, where it expels polar and ionic compounds into the bile. MRP2 is also present in the apical membrane of enterocytes and epithelial cells of proximal tubules of the kidney. Inhibition of MRP2 transport can lead to the accumulation of metabolites and other MRP2 substrates up to toxic levels in these cells. The transport properties of MRP2 are frequently studied with the vesicular transport assay. The assay identifies compounds that interact with MRP2 by measuring the effect of a compound on the transport of a radioactively labeled or fluorescent probe. We have compared the effect of eight selected test compounds (quercetin, disopyramide, paracetamol, indomethacin, diclofenac, estrone-3-sulfate, budesonide, and thioridazine) on the MRP2-mediated transport of three commonly used probes: 5(6)-carboxy-2,7-dichlorofluorescein, leukotriene C4 and estradiol-17-β-d-glucuronide (E217βG). Five of the test compounds had different probe-dependent effects on the MRP2-mediated transport, suggesting differences in the transport mechanism of the probes. Our results underline the complexity of substrate recognition by these efflux transporters and the difficulties in directly comparing results obtained with different assays, especially when different probes are used.

Topics: Acetaminophen; Animals; Baculoviridae; Biological Assay; Biological Transport; Budesonide; Cell Membrane; Cells, Cultured; Diclofenac; Disopyramide; Estradiol; Estrone; Fluoresceins; Genetic Vectors; Humans; Indomethacin; Inhibitory Concentration 50; Leukotriene C4; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Quercetin; Recombinant Proteins; Spodoptera; Statistics as Topic; Thioridazine; Transfection

Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (ΔMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) ΔMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of ΔMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of ΔMRP1 that traffics to the plasma membrane and transports leukotriene C(4), 17β-estradiol 17-β-D-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.

Topics: Alanine; Amino Acid Sequence; Amino Acid Substitution; Biological Transport; Cell Line, Transformed; Cell Membrane; Cysteine; Estradiol; Estrone; HEK293 Cells; Humans; Kinetics; Leukotriene C4; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; Structure-Activity Relationship

Although pharmaceutical excipients are supposed to be pharmacologically inactive, solubilizing agents like Cremophor EL have been shown to interact with cytochrome P450 (CYP)-dependent drug metabolism as well as efflux transporters such as P-glycoprotein (ABCB1) and multidrug resistance associated protein 2 (ABCC2). However, knowledge about their influence on the function of uptake transporters important in drug disposition is very limited. In this study we investigated the in vitro influence of polyethylene glycol 400 (PEG), hydroxypropyl-β-cyclodextrin (HPCD), Solutol HS 15 (SOL), and Cremophor EL (CrEL) on the organic anion transporting polypeptides (OATP) 1A2, OATP2B1, OATP1B1, and OATP1B3 and the Na(+)/taurocholate cotransporting polypeptide (NTCP). In stably transfected human embryonic kidney cells we analyzed the competition of the excipients with the uptake of bromosulfophthalein in OATP1B1, OATP1B3, OATP2B1, and NTCP, estrone-3-sulfate (E(3)S) in OATP1A2, OATP1B1, and OATP2B1, estradiol-17β-glucuronide in OATP1B3, and taurocholate (TA) in OATP1A2 and NTCP cells. SOL and CrEL were the most potent inhibitors of all transporters with the strongest effect on OATP1A2, OATP1B3, and OATP2B1 (IC(50) < 0.01%). HPCD also strongly inhibited all transport proteins but only for substrates containing a sterane-backbone. Finally, PEG seems to be a selective and potent modulator of OATP1A2 with IC(50) values of 0.05% (TA) and 0.14% (E(3)S). In conclusion, frequently used solubilizing agents were shown to interact substantially with intestinal and hepatic uptake transporters which should be considered in drug development. However, the clinical relevance of these findings needs to be evaluated in further in vivo studies.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; beta-Cyclodextrins; Biological Transport; Cell Line; Estradiol; Estrone; Excipients; Glycerol; HEK293 Cells; Humans; Multidrug Resistance-Associated Protein 2; Organic Anion Transporters; Organic Anion Transporters, Sodium-Dependent; Polyethylene Glycols; Stearic Acids; Sulfobromophthalein; Symporters; Taurocholic Acid

Organic anion-transporting polypeptides (OATPs) are multispecific transporters that mediate the uptake of numerous drugs and xenobiotics into cells. Here, we examined the effect of green tea (Camellia sinensis) catechins on the function of the four OATPs expressed in human enterocytes and hepatocytes. Uptake of the model substrate estrone-3-sulfate by cells expressing OATP1A2, OATP1B1, OATP1B3, or OATP2B1 was measured in the absence and presence of the four most abundant flavonols found in green tea. Uptake by OATP1A2, OATP1B1, and OATP2B1 was inhibited by epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) in a concentration-dependent way. In contrast, OATP1B3-mediated uptake of estrone-3-sulfate was strongly stimulated by EGCG at low substrate concentrations. The effect of EGCG on OATP1B3 was also studied with additional substrates: uptake of estradiol-17β-glucuronide was unchanged, whereas uptake of Fluo-3 was noncompetitively inhibited. Both ECG and EGCG were found to be substrates of OATP1A2 (K(m) values of 10.4 and 18.8 μM, respectively) and OATP1B3 (34.1 and 13.2 μM, respectively) but not of OATP1B1 or OATP2B1. These results indicate that two of the major flavonols found in green tea have a substantial effect on the function of OATPs expressed in enterocytes and hepatocytes and can potentially alter the pharmacokinetics of drugs and other OATP substrates. In addition, the diverse effects of EGCG on the transport of other OATP1B3 substrates suggest that different transport/binding sites are involved.

Topics: Aniline Compounds; Animals; Antioxidants; Biological Transport; Camellia sinensis; Catechin; Cell Line; CHO Cells; Cricetinae; Cricetulus; Drug Interactions; Enterocytes; Estradiol; Estrone; Fluorescent Dyes; Hepatocytes; Humans; Organic Anion Transporters; Tea; Xanthenes

Organic anion-transporting polypeptides (Oatp) 1a1 and 1a4 were deleted by homologous recombination, and mice were characterized for Oatp expression in liver and kidney, transport in isolated hepatocytes, in vivo disposition of substrates, and urinary metabolomic profiles. Oatp1a1 and Oatp1a4 proteins were undetected in liver, and both lines were viable and fertile. Hepatic constitutive messenger RNAs (mRNAs) for Oatp1a4, 1b2, or 2b1 were unchanged in Oatp1a1⁻/⁻ mice, whereas renal Oatp1a4 mRNA decreased approximately 50% (both sexes). In Oatp1a4⁻/⁻ mice, no changes in constitutive mRNAs for other Oatps were observed. Uptake of estradiol-17β-D-glucuronide and estrone-3-sulfate in primary hepatocytes decreased 95 and 75%, respectively, in Oatp1a1⁻/⁻ mice and by 60 and 30%, respectively, in Oatp1a4⁻/⁻ mice. Taurocholate uptake decreased by 20 and 50% in Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, respectively, whereas digoxin was unaffected. Plasma area under the curve (AUC) for estradiol-17β-D-glucuronide increased 35 and 55% in male and female Oatp1a1⁻/⁻ mice, respectively, with a concurrent 50% reduction in liver-to-plasma ratios. In contrast, plasma AUC or tissue concentrations of estradiol-17β-D-glucuronide were unchanged in Oatp1a4⁻/⁻ mice. Plasma AUCs for dibromosulfophthalein increased nearly threefold in male Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, increased by 40% in female Oatp1a4⁻/⁻ mice, and were unchanged in female Oatp1a1⁻/⁻ mice. In both lines, no changes in serum ALT, bilirubin, and cholesterol were noted. NMR analyses showed no generalized increase in urinary excretion of organic anions. However, urinary excretion of taurine decreased by 30-40% and was accompanied by increased excretion of isethionic acid, a taurine metabolite generated by intestinal bacteria, suggesting some perturbations in intestinal bacteria distribution.

Topics: Animals; Area Under Curve; Biological Transport; Blotting, Western; Estradiol; Estrone; Female; Gene Deletion; Hepatocytes; Homologous Recombination; Isethionic Acid; Kidney; Liver; Male; Metabolomics; Mice; Mice, Knockout; Organic Anion Transporters; RNA, Messenger; Taurine; Taurocholic Acid

While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes. In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 μM) was 27.5 (±15.0) μl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17β-D-glucuronide (E(2)-17β-G; 1 μM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6) μl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 μM) significantly (p<0.01) decreased the accumulation of estrone-3-sulfate and E(2)-17β-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) μl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 μM) and the OCT3 inhibitor corticosterone (1 μM) resulted in a significant (p<0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation (R(2)=0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.

Topics: 1-Methyl-4-phenylpyridinium; Adolescent; Adult; Biological Transport; Corticosterone; Cryopreservation; Digoxin; Estradiol; Estrone; Hepatocytes; Humans; Male; Membrane Transport Proteins; Middle Aged; Organic Anion Transporters; Organic Anion Transporters, Sodium-Dependent; Organic Anion Transporters, Sodium-Independent; Organic Cation Transport Proteins; Organic Cation Transporter 1; Prazosin; Rifampin; RNA, Messenger; Solute Carrier Organic Anion Transporter Family Member 1B3; Symporters; Taurocholic Acid

Organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are liver-specific transporters that mediate the uptake of a broad range of drugs into hepatocytes, including statins, antibiotics, and many anticancer drugs. Compounds that alter transport by one or both of these OATPs could potentially be used to target drugs to hepatocytes or improve the bioavailability of drugs that are cleared by the liver. In this study, we applied a bioassay-guided isolation approach to identify such compounds from the organic extract of Rollinia emarginata Schlecht (Annonaceae). Fractions of the plant extract were screened for effects on OATP1B1- and OATP1B3-mediated transport of the model substrates estradiol-17β-glucuronide and estrone-3-sulfate. We isolated three compounds, ursolic acid, oleanolic acid, and 8-trans-p-coumaroyloxy-α-terpineol, which inhibited estradiol-17β-glucuronide uptake by OATP1B1 but not OATP1B3. In addition, a rare compound, quercetin 3-O-α-l-arabinopyranosyl(1→2) α-L-rhamnopyranoside, was identified that had distinct effects on each OATP. OATP1B1 was strongly inhibited, as was OATP1B3-mediated transport of estradiol-17β-glucuronide. However, OATP1B3-mediated uptake of estrone-3-sulfate was stimulated 4- to 5-fold. Kinetic analysis of this stimulation revealed that the apparent affinity for estrone-3-sulfate was increased (decreased K(m)), whereas the maximal rate of transport (V(max)) was significantly reduced. These results demonstrate a mechanism through which the hepatic uptake of drug OATP substrates could be stimulated.

Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Drug Discovery; Estradiol; Estrone; Hepatocytes; Kinetics; Liver; Liver-Specific Organic Anion Transporter 1; Molecular Targeted Therapy; Oleanolic Acid; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Plant Bark; Plant Extracts; Rollinia; Solute Carrier Organic Anion Transporter Family Member 1B3; Terpenes; Triterpenes; Ursolic Acid

The human liver-specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are involved in the elimination of numerous xenobiotics and drugs. Although dogs are frequently used for toxicologic and pharmacokinetic characterization of novel drugs, nothing is known about their OATP1B1/1B3 ortholog. Therefore, we cloned and characterized the first canine organic anion transporting polypeptide from dog liver, termed Oatp1b4. The isolated Oatp1b4 cDNA comprises 3661 base pairs (bp) with an open reading frame of 2076bp, encoding a 692-amino acid protein with a molecular mass of approximately 85kDa. The Oatp1b4 gene is approximately 61kb long and has a similar organization as the human OATP1B1 and OATP1B3 with 13 exons identical in length. Northern blot analysis shows that Oatp1b4 is predominantly expressed in the liver. Oatp1b4 mediates sodium-independent transport of typical organic anions including bromosulfophthalein (BSP), [D-penicillamine(2,5)]enkephalin (DPDPE), estradiol-17beta-glucuronide (E17betaG), estrone-3-sulfate and taurocholate. In addition, Oatp1b4 transports the OATP1B3-specific substrate cholecystokinin octapeptide (CCK-8). Kinetic studies showed that Oatp1b4-mediated E17betaG and estrone-3-sulfate transports were monophasic with K(m) values of 5+/-1microM and 33+/-4microM, respectively. In conclusion, the cloned canine Oatp1b4 will provide additional molecular basis to further characterize the species difference of the OATP1B family members.

Topics: Amino Acid Sequence; Animals; Base Pairing; Base Sequence; Biological Transport; Cell Line; Cloning, Molecular; DNA, Complementary; Dogs; Enkephalin, D-Penicillamine (2,5)-; Estradiol; Estrone; Exons; Genes; Humans; Introns; Kidney; Kinetics; Liver; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Sincalide; Substrate Specificity; Sulfobromophthalein; Taurocholic Acid

Polybrominated diphenyl ethers (PBDEs) are flame-retardants that upon chronic exposure enter the liver where they are biotransformed to potentially toxic metabolites. The mechanism by which PBDEs enter the liver is not known. However, due to their large molecular weights (MWs approximately 485 to 1000 Da), they cannot enter hepatocytes by simple diffusion. Organic anion-transporting polypeptides (OATPs) are responsible for hepatic uptake of a variety of amphipathic compounds of MWs larger than 350 Da. Therefore, in the present study, Chinese hamster ovary cell lines expressing OATP1B1, OATP1B3, and OATP2B1 were used to test the hypothesis that OATPs expressed in human hepatocytes would be responsible for the uptake of PBDE congeners 47, 99, and 153. The results demonstrated that PBDE congeners inhibited OATP1B1- and OATP1B3-mediated uptake of estradiol-17-beta-glucuronide as well as OATP2B1-mediated uptake of estrone-3-sulfate in a concentration-dependent manner. Direct uptake studies confirmed that all three PBDE congeners are substrates for the three tested hepatic OATPs. Detailed kinetic analysis revealed that OATP1B1 transported 2,2',4,4'-tetrabromodiphenyl ether (BDE47) with the highest affinity (K(m) = 0.31 microM) followed by 2,2',4,4',5-pentabromodiphenyl ether (BDE99) (K(m) = 0.91 microM) and 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE153) (K(m) = 1.91 microM). For OATP1B3, the order was the same (BDE47: K(m) = 0.41 microM; BDE99: K(m) = 0.70 microM; BDE153: K(m) = 1.66 microM), while OATP2B1 transported all three congeners with similar affinities (BDE47: K(m) = 0.81 microM; BDE99: K(m) = 0.87 microM; BDE153: K(m) = 0.65 microM). These results clearly suggest that uptake of PBDEs via these OATPs is a mechanism responsible for liver-specific accumulation of PBDEs.

Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Estradiol; Estrone; Flame Retardants; Halogenated Diphenyl Ethers; Hepatocytes; Humans; Liver; Organic Anion Transporters

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are responsible for the hepatic uptake of organic anions. They share similar sequences and structures with 12 putative transmembrane domains (TMs). Their substrate specificities are very broad and overlap each other, whereas each transporter specifically recognizes certain substrates. Because the homology of the amino acid sequence in the latter part of OATP1B1 and OATP1B3 is relatively low, to determine which TMs in the latter part of OATP1B1 are important for its substrate recognition, we constructed several cell lines expressing chimeric transporters in which some TMs of OATP1B1 were substituted with those of OATP1B3, and we investigated the transport kinetics of estrone-3-sulfate (E-sul; a substrate preferentially accepted by OATP1B1) and estradiol-17beta-D-glucuronide (EG; a substrate accepted by both transporters). As the number of substituted TMs at the N terminus with those of OATP1B3 increased, the K(m) value of E-sul greatly increased and its uptake clearance decreased. The substitution of TM7 or TM9 of OATP1B1 with that of OATP1B3 (named 1B1-TM7 or 1B1-TM9) did not change the transport kinetics of EG, whereas the K(m) value of E-sul in 1B1-TM9 increased 7.4-fold. Conversely, the substitution of TM8 resulted in an 18-fold increase in the K(m) value of E-sul and abolished the transporter-mediated uptake of EG. These results suggest that TM8 in OATP1B1 is critical for the substrate recognition of both E-sul and EG and that TM9 is important for the recognition of E-sul, whereas it is interchangeable with that of OATP1B3 for EG transport.

Topics: Biological Transport; Cell Line; Estradiol; Estrone; Humans; Kinetics; Liver-Specific Organic Anion Transporter 1; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Protein Structure, Tertiary; Solute Carrier Organic Anion Transporter Family Member 1B3; Substrate Specificity

The chemical space of registered oral drugs was explored for inhibitors of the human multidrug-resistance associated protein 2 (MRP2; ABCC2), using a data set of 191 structurally diverse drugs and drug-like compounds. The data set included a new reference set of 75 compounds, for studies of hepatic drug interactions with transport proteins, CYP enzymes, and compounds associated with liver toxicity. The inhibition of MRP2-mediated transport of estradiol-17beta-D-glucuronide was studied in inverted membrane vesicles from Sf9 cells overexpressing human MRP2. A total of 27 previously unknown MRP2 inhibitors were identified, and the results indicate an overlapping but narrower inhibitor space for MRP2 compared with the two other major ABC efflux transporters P-gp (ABCB1) and BCRP (ABCG2). In addition, 13 compounds were shown to stimulate the transport of estradiol-17beta-D-glucuronide. The experimental results were used to develop a computational model able to discriminate inhibitors from noninhibitors according to their molecular structure, resulting in a predictive power of 86% for the training set and 72% for the test set. The inhibitors were in general larger and more lipophilic and presented a higher aromaticity than the noninhibitors. The developed computational model is applicable in an early stage of the drug discovery process and is proposed as a tool for prediction of MRP2-mediated hepatic drug interactions and toxicity.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Antipsychotic Agents; Antiviral Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Cell Line; Computer Simulation; Cytochrome P-450 Enzyme System; Drug-Related Side Effects and Adverse Reactions; Estradiol; Humans; Insecta; Liver; Models, Molecular; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Pharmaceutical Preparations; Pharmacology; Structure-Activity Relationship

Multidrug resistance proteins (MRPs) are members of the "C" branch of the ATP-binding cassette transporter superfamily. Human MRP1 transports a wide range of natural product drugs and structurally diverse conjugated and unconjugated organic anions. Its closest relative is MRP3. Despite their structural similarity, the homologs differ substantially in their substrate specificity. It is noteworthy that MRP1 transports glutathione (GSH) and GSH conjugates and displays GSH-stimulated transport of a number of unconjugated and conjugated compounds. In contrast, MRP3 does not transport GSH and is a poor transporter of GSH conjugates. However, both proteins transport glucuronide conjugates, such as 17beta-estradiol 17-(beta-D-glucuronide). We have constructed a series of MRP1/MRP3 hybrids and used them to identify a region of MRP1 that is critical for binding and transport of GSH conjugates such as leukotriene C(4) (LTC(4)). Substitution of this region encompassing transmembrane helices 8 and 9 and portions of cytoplasmic loops 4 and 5 of MRP1 with the equivalent region of MRP3 eliminated LTC(4) transport. Transport of other substrates was either unaffected or enhanced. We identified three residues in this region: Tyr(440), Ile(441), and Met(443), mutation of which differentially affected transport. It is noteworthy that substitution of Tyr(440) with Phe, as found in MRP3, reduced LTC(4) and GSH-stimulated estrone-3-sulfate transport without affecting transport of other substrates tested. The mutation increased the K(m) for LTC(4) 5-fold and substantially reduced photolabeling of MRP1 by both [3H]LTC(4) and the GSH derivative, azidophenacyl-[35S]GSH. These results suggest that Tyr(440) makes a major contribution to recognition of GSH and the GSH moiety of conjugates such as LTC(4).

Topics: Amino Acid Sequence; Amino Acid Substitution; Cell Line; Cell Membrane; Cell Survival; Doxorubicin; Drug Resistance, Neoplasm; Estradiol; Estrone; Etoposide; Glutathione; Humans; Kinetics; Leukotriene C4; Methotrexate; Models, Molecular; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; Mutagenesis, Site-Directed; Protein Binding; Recombinant Fusion Proteins; Substrate Specificity; Transfection; Vincristine

The pregnane X receptor is a ligand-activated transcription factor that is abundantly expressed in hepatocytes. Numerous drugs are pregnane X receptor ligands. To bind to their receptor they must cross the sinusoidal membrane. Organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are polyspecific transporters expressed at the sinusoidal membrane of human hepatocytes. They mediate transport of a variety of drugs including the pregnane X receptor ligands rifampicin and dexamethasone. To test whether additional pregnane X receptor ligands interact with OATP1B1- and 1B3-mediated transport, we developed Chinese Hamster Ovary (CHO) cell lines stably expressing OATP1B1 or 1B3 at high levels. OATP1B1- and 1B3-mediated estradiol-17beta-glucuronide uptake was inhibited by several pregnane X receptor ligands in a concentration dependent way. IC(50) values for rifampicin, paclitaxel, mifepristone, and troglitazone were within their respective pharmacological free plasma concentrations. Kinetic analysis revealed that clotrimazole inhibits OATP1B1-mediated estradiol-17beta-glucuronide transport with a K(i) of 7.7+/-0.3 microM in a competitive way. However, uptake of OATP1B3-mediated estradiol-17beta-glucuronide was stimulated and this stimulation was due to an increased apparent affinity. Transport of estrone-3-sulfate was hardly affected while all other substrates tested were inhibited. Additional azoles like fluconazole, ketoconazole and miconazole did not stimulate OATP1B3-mediated estradiol-17beta-glucuronide transport. In summary, these results demonstrate that pregnane X receptor ligands, by inhibiting or stimulating OATP-mediated uptake, can lead to drug-drug interactions at the transporter level.

Topics: Animals; CHO Cells; Chromans; Clotrimazole; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Drug Interactions; Estradiol; Estrone; Humans; Kinetics; Ligands; Liver-Specific Organic Anion Transporter 1; Mifepristone; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Paclitaxel; Pregnane X Receptor; Receptors, Steroid; Rifampin; Solute Carrier Organic Anion Transporter Family Member 1B3; Thiazolidinediones; Transfection; Troglitazone; Xenobiotics

The human placenta has both protective and nurturing functions for the fetal organism. Uptake and elimination of xenobiotics and endogenous substances are facilitated by various transport proteins from the solute carrier (SLC) and ABC families, respectively. A functional interaction of uptake and elimination, which is a prerequisite for vectorial transport across cellular barriers, has not been described for placenta. In this study, we examined expression of organic anion transporter (OAT) 4 (SLC22A11), organic anion transporting polypeptide (OATP) 2B1 (SLCO2B1, OATP-B), and breast cancer resistance protein (BCRP) (ABCG2) in human placenta (n = 71) because all three proteins are involved in transmembranal transfer of estrone 3 sulfate (E3S; metabolic product) and dehydroepiandrosterone sulfate (DHEAS; precursor molecule). On the mRNA level, we found a significant correlation of OATP2B1 and BCRP (R(2) = 0.534; p < 0.01) but not between OAT4 and BCRP (R(2) = -0.104; p > 0.05). Localization studies confirmed basal expression of OATP2B1 and apical expression of BCRP. To study functional interactions between OATP2B1 and BCRP, we developed a Madin-Darby canine kidney cell model expressing both transport proteins simultaneously (OATP2B1 and BCRP in the basal and apical membrane, respectively). Using this cell model in a transwell system resulted in a significantly increased basal to apical transport of both E3S and DHEAS, when both transporters were expressed with no change of transfer in the apical to basal direction. Taken together, these data show the potential for a functional interaction of OATP2B1 and BCRP in transepithelial transport of steroid sulfates in human placenta.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Cell Line; Cell Membrane; Dehydroepiandrosterone Sulfate; Dogs; Estradiol; Estrone; Female; Humans; Neoplasm Proteins; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Placenta; RNA, Messenger

The elimination process of the endothelin receptor antagonist bosentan (Tracleer) in humans is entirely dependent on metabolism mediated by two cytochrome P450 (P450) enzymes, i.e., CYP3A4 and CYP2C9. Most interactions with concomitantly administered drugs can be rationalized in terms of inhibition of these P450 enzymes. The increased bosentan concentrations observed in the presence of cyclosporin A, rifampicin, or sildenafil, however, are incompatible with this paradigm and prompted the search for alternative mechanisms governing these interactions. In the present article, we identify bosentan and its active plasma metabolite, Ro 48-5033 (4-(2-hydroxy-1,1-dimethyl-ethyl)-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-[2,2']bipyrimidinyl-4-yl]-benzenesulfonamide), as substrates of the human organic anion transporting polypeptides (OATP) OATP1B1 and OATP1B3. Bosentan uptake into Chinese hamster ovary cells expressing these OATP transporters was efficiently inhibited by cyclosporin A and rifampicin with IC(50) values significantly below their effective plasma concentrations in humans. The phosphodiesterase-5 inhibitor sildenafil was also shown to interfere with OATP-mediated transport, however, at concentrations above those achieved in therapeutic use. Therefore, inhibition of bosentan hepatic uptake may represent an alternative/complementary mechanism to rationalize some of the pharmacokinetic interactions seen in therapeutic use. A similar picture has been drawn for drugs like pitavastatin and fexofenadine, drugs that are mainly excreted in unchanged form. Bosentan elimination, in contrast, is entirely dependent on metabolism. Therefore, the described interactions with rifampicin, cyclosporin A, and, to a lesser extent, sildenafil represent evidence that inhibition of hepatic uptake may become the rate-limiting step in the overall elimination process even for drugs whose elimination is entirely dependent on metabolism.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Biological Transport; Bosentan; CHO Cells; Cricetinae; Cricetulus; Cyclosporine; Cytochrome P-450 CYP2C9; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dehydroepiandrosterone Sulfate; Drug Interactions; Enzyme Inhibitors; Estradiol; Estrone; Humans; Liver-Specific Organic Anion Transporter 1; Molecular Structure; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Piperazines; Purines; Pyrimidines; Rifampin; Sildenafil Citrate; Solute Carrier Organic Anion Transporter Family Member 1B3; Sulfonamides; Sulfones; Warfarin

It has already been demonstrated that pitavastatin, a novel potent HMG-coenzyme A reductase inhibitor, is taken up into human hepatocytes mainly by organic anion transporting polypeptide (OATP) 1B1. Because OATP2B1 is also localized in the basolateral membrane of human liver, we took two approaches to further confirm the minor contribution of OATP2B1 to the hepatic uptake of pitavastatin. Western blot analysis revealed that the ratio of the band density of OATP2B1 in human hepatocytes to that in our expression system is at least 6-fold lower compared with OATP1B1 and OATP1B3. The uptake of pitavastatin in human hepatocytes could be inhibited by both estrone-3-sulfate (OATP1B1/OATP2B1 inhibitor) and estradiol-17beta-D-glucuronide (OATP1B1/OATP1B3 inhibitor). These results further supported the idea that OATP1B1 is a predominant transporter for the hepatic uptake of pitavastatin. Then, to explore the possibility of OATP1B1-mediated drug-drug interaction, we checked the inhibitory effects of various drugs on the pitavastatin uptake in OATP1B1-expressing cells and evaluated whether the in vitro inhibition was clinically significant or not. As we previously reported, we used the methodology for estimating the maximum unbound concentration of inhibitors at the inlet to the liver (I(u,in,max)). Judging from I(u,in,max) and inhibition constant (K(i)) for OATP1B1, several drugs (especially cyclosporin A, rifampicin, rifamycin SV, clarithromycin, and indinavir) have potentials for interacting with OATP1B1-mediated uptake of pitavastatin. The in vitro experiments could support the clinically observed drug-drug interaction between pitavastatin and cyclosporin A. These results suggest that we should pay attention to the concomitant use of some drugs with pitavastatin.

Topics: Cell Line; Cyclosporine; Drug Interactions; Estradiol; Estrone; Hepatocytes; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kinetics; Liver-Specific Organic Anion Transporter 1; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Quinolines; Solute Carrier Organic Anion Transporter Family Member 1B3; Transfection

Telmisartan, a nonpeptide angiotensin II receptor antagonist, is selectively distributed to liver. In the present study, we have characterized the contribution of organic anion transporting polypeptide (OATP) isoforms to the hepatic uptake of telmisartan by isolated rat hepatocytes, human cryopreserved hepatocytes, and human transporter-expressing cells. Because it is difficult to evaluate the transport activity of telmisartan because of its extensive adsorption to cells and culture materials, we performed the uptake study in the presence of human serum albumin. The saturable uptake of telmisartan into isolated rat hepatocytes took place in a Na(+)-independent manner and was inhibited by pravastatin, taurocholate, and digoxin, which are Oatp substrates and inhibitors, but not by organic cation, tetraethylammonium, indicating the involvement of Oatp isoforms in its uptake into rat hepatocytes. To identify which human OATP transporters are important for the hepatic uptake of telmisartan, the uptake assay was carried out using OATP1B1- and OATP1B3-expressing human embryonic kidney 293 cells and cryopreserved human hepatocytes. The uptake of telmisartan by OATP1B3-expressing cells was saturable (K(m) = 0.81 microM) and significantly higher than that by vector-transfected cells. In contrast, no significant uptake was observed in OATP1B1-expressing cells. We also observed the saturable uptake of telmisartan by human hepatocytes. Thirty micromolar estrone-3-sulfate, which can selectively inhibit OATP1B1-mediated uptake compared with OATP1B3, did not inhibit the uptake of telmisartan in human hepatocytes, whereas it could inhibit the uptake of estradiol 17beta-d-glucuronide mediated by OATP1B1. These results suggest that OATP1B3 is predominantly involved in the hepatic uptake of telmisartan in humans.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Benzoates; Cell Line; Estradiol; Estrone; Hepatocytes; Humans; Liver-Specific Organic Anion Transporter 1; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Rats; Rats, Sprague-Dawley; Serum Albumin; Sincalide; Solute Carrier Organic Anion Transporter Family Member 1B3; Telmisartan; Transfection

We previously determined that expression of human multidrug resistance protein (MRP) 8, a recently described member of the MRP family of ATP-binding cassette transporters, enhances cellular extrusion of cyclic nucleotides and confers resistance to nucleotide analogs (J Biol Chem 278:29509-29514, 2003). However, the in vitro transport characteristics of the pump have not been determined. In this study, the substrate selectivity and biochemical activity of MRP8 is investigated using membrane vesicles prepared from LLC-PK1 cells transfected with MRP8 expression vector. Expression of MRP8 is shown to stimulate the ATP-dependent uptake of a range of physiological and synthetic lipophilic anions, including the glutathione S-conjugates leukotriene C4 and dinitrophenyl S-glutathione, steroid sulfates such as dehydroepiandrosterone 3-sulfate (DHEAS) and estrone 3-sulfate, glucuronides such as estradiol 17-beta-D-glucuronide (E(2)17betaG), the monoanionic bile acids glycocholate and taurocholate, and methotrexate. In addition, MRP8 is competent in the in vitro transport of cAMP and cGMP, in accord with the results of our previously reported cellular studies. DHEAS, E(2)17betaG, and methotrexate were transported with K(m) and V(max) values of 13.0 +/- 0.8 microM and 34.9 +/- 9.5 pmol/mg/min, 62.9 +/- 12 microM and 62.0 +/- 5.2 pmol/mg/min, and 957 +/- 28 microM and 317 +/- 17 pmol/mg/min, respectively. Based upon the stimulatory action of DHEAS on uptake of E(2)17betaG, the attenuation of this effect at high DHEAS concentrations and the lack of reciprocal promotion of DHEAS uptake by E(2)17betaG, a model involving nonreciprocal constructive interactions between some transport substrates is invoked. These results suggest that MRP8 participates in physiological processes involving bile acids, conjugated steroids, and cyclic nucleotides and indicate that the pump has complex interactions with its substrates.

Topics: Animals; ATP-Binding Cassette Transporters; Bile Acids and Salts; Dose-Response Relationship, Drug; Estradiol; Humans; Leukotriene C4; LLC-PK1 Cells; Protein Transport; Swine

Fexofenadine hydrochloride (FEX), a second generation H(1)-receptor antagonist, is mainly eliminated from the liver into bile in unchanged form. Recent studies have shown that FEX can be accepted by human MDR1 (P-glycoprotein), OATP1A2 [organic anion-transporting polypeptide (OATP)-A, and OATP2B1 (OATP-B)] expression systems. However, other transporters responsible for the hepatic uptake of FEX have not yet been identified. In the present study, we evaluated the contribution of OATP family transporters, namely OATP1B1 (OATP2/OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), to FEX uptake using transporter-expressing HEK293 (human embryonic kidney) cells. The uptake of FEX in OATP1B3-expressing cells was significantly greater than that in vector-transfected cells. On the other hand, OATP1B1- or OATP2B1-mediated uptake of FEX was not statistically significant. OATP1B3-mediated transport could be explained by a one-saturable component with a Michaelis constant (K(m)) of 108 +/- 11 microM. The inhibitory effect of FEX on the uptake of estrone-3-sulfate (E(1)S), cholecystokinin octapeptide (CCK-8), and 17beta-estradiol-17beta-d-glucuronide (E(2)17betaG) was also examined. Both OATP1B1- and OATP1B3-mediated E(2)17betaG uptake was inhibited by FEX. The K(i) values were 148 +/- 61 and 205 +/- 72 microM for OATP1B1 and OATP1B3, respectively. FEX also inhibited OATP1B3-mediated CCK-8 uptake and OATP1B1-mediated E(1)S uptake with a K(i) value of 83.3 +/- 15.3 and 257 +/- 84 microM, respectively, suggesting that FEX could not be used as a specific inhibitor for OATP1B1 and OATP1B3, although FEX was preferentially accepted by OATP1B3. In conclusion, this is, to our knowledge, the first demonstration that OATP1B3 is thought to be a major transporter involved in hepatic uptake of FEX in humans.

Topics: Cell Line; Estradiol; Estrone; Histamine H1 Antagonists; Humans; Liver; Liver-Specific Organic Anion Transporter 1; Organic Anion Transport Protein 1; Sincalide; Terfenadine

The present study was aimed at investigating the involvement of mouse organic anion transporting polypeptide 14 (mOatp14) in the uptake of T4 across the blood-brain barrier. Functional expression of mOatp14 in HEK293 cells revealed that T4 and rT3 are high affinity substrates of mOatp14 (Michaelis constant, 0.34 and 0.46 microm, respectively), and the specific uptake of T3 was 4-fold less than that of T4 and rT3. Taurocholate, probenecid, and estrone-3-sulfate were moderate inhibitors for mOatp14, whereas digoxin (substrate of Oatp2), benzylpenicillin (substrate of Oat3), and large neutral amino acids had no effect. mOatp14 is widely expressed throughout the brain, except for the cerebellum. The expression of mOatp14 in the isolated brain capillaries and the choroid plexus was shown by Western blot. The uptake clearance of T4 by the cerebral cortex determined using the in situ brain perfusion technique in mice was 580 microl/min.g tissue, 3-fold greater than that by the cerebellum, and a saturable component (Michaelis constant, 1.0 microm) accounts for the major fraction of the total uptake. Taurocholate inhibited the uptake of T4 by the cerebral cortex completely, but the inhibition by estrone-3-sulfate was partial (50%). These results suggest that transporters play a predominant role in the delivery of T4 to the brain, and mOatp14 accounts for estrone-3-sulfate inhibitable fraction, at least partly. The absence of inhibition by digoxin, benzylpenicillin, leucine, and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid for the uptake of T4 by the cerebral cortex suggests the presence of other unknown transporter for T4 uptake by the brain. Immunohistochemical staining revealed basolateral localization of mOatp14 in the choroid plexus in which it may also play a role in T4 uptake.

Topics: Animals; Biological Transport; Blood-Brain Barrier; Cerebellum; Cerebral Cortex; Choroid Plexus; Estradiol; Iodine Radioisotopes; Male; Mice; Organic Cation Transport Proteins; Thyroxine; Triiodothyronine

We describe the cloning, functional characterization and tissue localization of a novel membrane transporter of the OATP/Oatp-gene family obtained from liver and kidney of cattle (Bos taurus). The carrier protein exhibits highest sequence identity to the human OATP1A2 (previously called OATP-A) and is, therefore, named bovine Oatp1a2. Bovine Oatp1a2 received the gene symbol Slco1a2 that is identical to the SLC classification of human OATP1A2 (SLCO1A2, previously called SLC21A3) and is likely an orthologue of the human gene. Two different full-length bOatp1a2 cDNAs of 2316-bp and 3504-bp were obtained and encoded for a 666 amino acid membrane protein, which contains twelve putative transmembrane spanning domains. Bovine Oatp1a2 expression was detected in liver, kidney, brain and adrenal gland. Uptake studies in cRNA-injected oocytes demonstrated that bOatp1a2 transports estrone-3-sulfate and taurocholate, with K(m) values of 9.6 microM and 51 microM, respectively, and estradiol-17beta-glucuronide. However, the structurally-related heart glycosides ouabain (1 microM) and digoxin (1 microM) are neither transported by bovine Oatp1a2 nor by human OATP1A2. We conclude that based on the tested substrates bovine Oatp1a2 shows functional homology to human OATP1A2.

Topics: Animals; Base Sequence; Biological Transport; Cattle; Cloning, Molecular; DNA, Complementary; Estradiol; Estrone; Kinetics; Membrane Proteins; Molecular Sequence Data; Organic Anion Transporters; Sequence Alignment; Taurocholic Acid; Tissue Distribution

Multidrug resistance protein 1 (MRP1) transports a wide range of structurally diverse conjugated and nonconjugated organic anions and some peptides, including oxidized and reduced glutathione (GSH). The protein confers resistance to certain heavy metal oxyanions and a variety of natural product-type chemotherapeutic agents. Elevated levels of MRP1 have been detected in many human tumors, and the protein is a candidate therapeutic target for drug resistance reversing agents. Previously, we have shown that human MRP1 (hMRP1) and murine MRP1 (mMRP1) differ in their substrate specificity despite a high degree of structural conservation. Since rat models are widely used in the drug discovery and development stage, we have cloned and functionally characterized rat MRP1 (rMRP1). Like mMRP1 and in contrast to hMRP1, rMRP1 confers no, or very low, resistance to anthracyclines and transports the two estrogen conjugates, 17beta-estradiol-17-(beta-d-glucuronide) (E217betaG) and estrone 3-sulfate, relatively poorly. Mutational studies combined with vesicle transport assays identified several amino acids conserved between rat and mouse, but not hMRP1, that make major contributions to these differences in substrate specificity. Despite the fact that the rodent proteins transport E217betaG poorly and the GSH-stimulated transport of estrone 3-sulfate is low compared with hMRP1, site-directed mutagenesis studies indicate that different nonconserved amino acids are involved in the low efficiency with which each of the two estrogen conjugates is transported. Our studies also suggest that although rMRP1 and mMRP1 are 95% identical in primary structure, their substrate specificities may be influenced by amino acids that are not conserved between the two rodent proteins.

Topics: Amino Acid Sequence; Animals; Carrier Proteins; Cell Line; Cloning, Molecular; DNA, Complementary; Doxorubicin; Embryo, Mammalian; Estradiol; Estradiol Congeners; Estrone; Gene Expression Regulation; Humans; Kidney; Leukotriene C4; Mice; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; Mutagenesis, Site-Directed; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid; Species Specificity; Substrate Specificity; Transfection; Tritium

Transport of various amphipathic organic compounds is mediated by organic anion transporting polypeptides (OATPs in humans, Oatps in rodents), which belong to the solute carrier family 21A (SLC21A/Slc21a). Several of these transporters exhibit a broad and overlapping substrate specificity and are expressed in a variety of different tissues. We have isolated and functionally characterized OATP-F (SLC21A14), a novel member of the OATP family. The cDNA (3059 bp) contains an open reading frame of 2136 bp encoding a protein of 712 amino acids. Its gene containing 15 exons is located on chromosome 12p12. OATP-F exhibits 47-48% amino acid identity with OATP-A, OATP-C, and OATP8, the genes of which are clustered on chromosome 12p12. OATP-F is predominantly expressed in multiple brain regions and Leydig cells of the testis. OATP-F mediates high affinity transport of T(4) and reverse T(3) with apparent K(m) values of approximately 90 nM and 128 nM, respectively. Substrates less well transported by OATP-F include T(3), bromosulfophthalein, estrone-3-sulfate, and estradiol-17beta-glucuronide. Furthermore, OATP-F-mediated T(4) uptake could be cis-inhibited by L-T(4) and D-T(4), but not by 3,5-diiodothyronine, indicating that T(4) transport is not stereospecific, but that 3',5'-iodination is important for efficient transport by OATP-F. Thus, in contrast to most other family members, OATP-F has a more selective substrate preference and may play an important role in the disposition of thyroid hormones in brain and testis.

Topics: Amino Acid Sequence; Animals; Brain; CHO Cells; Chromosomes, Human, Pair 12; Cloning, Molecular; Cricetinae; Diiodothyronines; Estradiol; Estrone; Female; Humans; Leydig Cells; Male; Membrane Proteins; Molecular Sequence Data; Oocytes; Organ Specificity; Organic Anion Transporters; Sequence Homology, Amino Acid; Sulfobromophthalein; Testis; Thyroxine; Triiodothyronine; Xenopus

Unconjugated bilirubin (UCB) is currently believed to cross the placenta only by passive diffusion. To assess whether carrier-mediated transport might be involved, the uptake of [(3)H]-UCB by basal (bTPM) and apical (aTPM) plasma membrane vesicles from human placental trophoblast at term was investigated. In both types of vesicles, the uptake of [(3)H]-UCB into an osmotically sensitive space was temperature-dependent, independent of the presence of Na(+), and not affected by changes in membrane potential. The uptake of [(3)H]-UCB by aTPM, but not bTPM, was activated by ATP hydrolysis and inhibited by vanadate. Thus, the exact contribution of both inside out and right-side out bTPM to UCB uptake could not be distinguished, while only inverted aTPM were expected to carry out ATP-dependent UCB uptake. In bTPM and aTPM, uptake of free (unbound) [(3)H]-UCB (B(f)) consisted of a dominant, saturable, presumably carrier-mediated process and a diffusional component that became predominant only at B(f) near or above aqueous solubility limit for UCB (70 nM ). For bTPM, K(m)=7.2 nM; V(max)=9.8 pmol/20s/mg protein; and diffusion coefficient (K(D))=0.14 ml/20s/mg protein. For aTPM in the presence of 9.5m M ATP, K(m)=18 n M; V(max)=131 pmol/20s/mg protein; and K(D)=0.47 ml/20s/mg protein. The uptake of [(3)H]-UCB by bTPM was cis-inhibited by estrone-3-sulfate and estradiol-17 beta-glucuronide and trans-stimulated by unlabelled UCB and bromosulphopthalein. ATP-dependent UCB uptake by aTPM was cis-inhibited by doxorubicin, cholic acid, methotrexate and pronenecid. These findings suggest the presence of distinct transporters in the two domains of human placental trophoblast that could cooperate to transfer UCB from the foetus to the maternal circulation.

Topics: Adenosine Triphosphate; Bilirubin; Biological Transport; Carrier Proteins; Cell Membrane; Diffusion; Estradiol; Estrone; Female; Humans; Hydrolysis; Membrane Potentials; Osmolar Concentration; Pregnancy; Sulfobromophthalein; Temperature; Tritium; Trophoblasts

Human organic anion transporting polypeptide 2 (OATP2/SLC21A6) and multidrug resistance-associated protein 2 (MRP2/ABCC2) play important roles in the vectorial transport of organic anions across hepatocytes. In the present study, we have established a double-transfected Madin-Darby canine kidney (MDCK II) cell monolayer, which expresses both OATP2 and MRP2 on basal and apical membranes, respectively. The basal-to-apical transport of 17 beta estradiol 17 beta-d-glucuronide (E(2)17 beta G), pravastatin, and leukotriene C(4) (LTC(4)), which are substrates of OATP2 and MRP2, was significantly higher than that in the opposite direction in the double-transfected cells. Such vectorial transport was also observed for taurolithocholate sulfate, which is transported by rat oatp1 and Mrp2. The K(m) values of E(2)17 beta G and pravastatin for the basal-to-apical flux were 27.9 and 24.3 microm, respectively, which were comparable with those reported for OATP2. Moreover, the MRP2-mediated export of E(2)17 beta G across the apical membrane was not saturated. In contrast, basal-to-apical transport of estrone-3-sulfate and dehydroepiandrosterone sulfate, which are significantly transported by OATP2, but not by MRP2, was not stimulated by MRP2 expression. The double-transfected MDCK II monolayer expressing both OATP2 and MRP2 may be used to analyze the hepatic vectorial transport of organic anions and to screen the transport profiles of new drug candidates.

Topics: Animals; Biological Transport; Cell Culture Techniques; Cell Line; Cell Membrane; Dehydroepiandrosterone Sulfate; Dogs; Estradiol; Estrone; Humans; Kidney; Kinetics; Liver-Specific Organic Anion Transporter 1; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Rats; Recombinant Proteins; Taurolithocholic Acid; Transfection

To assess the functional characteristics of human organic anion transporter B (OATP-B) in comparison with those of the known, liver-specific OATP-C.. OATP-B or -C was expressed in HEK293 cells or Xenopus oocytes, and uptakes of estradiol-17beta-glucuronide and estrone-3-sulfate were measured using radiolabeled compounds.. OATP-C transported both estrone-3-sulfate and estradiol-17beta-glucuronide, whereas OATP-B transported only the former. OATP-C-mediated uptake of estrone-3-sulfate exhibited biphasic saturation kinetics, whereas transports of estradiol-17beta-glucuronide by OATP-C and estrone-3-sulfate by OATP-B followed single-saturation kinetics. Inhibition kinetics showed that only the high-affinity site for estrone-3-sulfate on OATP-C was shared with glucuronide conjugates. Uptake of [3H]estrone-3-sulfate by OATP-B was inhibited by sulfate conjugates but not by glucuronide conjugates, whereas its uptake by OATP-C was inhibited by both types of conjugates.. OATP-B accepted sulfate conjugates of steroids but not glucuronide conjugates, whereas OATP-C transported both types of steroid conjugates. Transport of estrone-3-sulfate by OATP-B and -C followed single- and biphasic-saturation kinetics, respectively, and the high-affinity site on OATP-C was the same as that for estradiol-17beta-glucuronide. Other OATPs, OATP-A and OATP-8, reportedly exhibit different preferences for steroid conjugates, and the specific recognition of sulfate conjugates seems to be unique to OATP-B.

Topics: Algorithms; Animals; Estradiol; Estrogens, Conjugated (USP); Estrone; Humans; Kinetics; Liver-Specific Organic Anion Transporter 1; Oocytes; Organic Anion Transporters; Reverse Transcriptase Polymerase Chain Reaction; Substrate Specificity; Xenopus

Murine multidrug resistance protein 1 (mrp1), unlike human MRP1, does not confer resistance to anthracyclines. Previously, we have shown that a human/murine hybrid protein containing amino acids 959-1187 of MRP1 can confer resistance to these drugs. We have now examined the functional characteristics of mutant proteins in which we have converted individual amino acids in the comparable region of mrp1 to those present at the respective locations in MRP1. These mutations had no effect on the drug resistance profile conferred by mrp1 with the exception of converting glutamine 1086 to glutamate, as it is in the corresponding position (1089) in MRP1. This mutation created a protein that conferred resistance to doxorubicin without affecting vincristine resistance, or the ability of mrp1 to transport leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG). Furthermore, mutation Q1086D conferred the same phenotype as mutation Q1086E while the mutation Q1086N did not detectably alter the drug resistance profile of mrp1, suggesting that an anionic side chain was required for anthracycline resistance. To confirm the importance of MRP1 E1089 for conferring resistance to anthracyclines, we mutated this residue to Gln, Asp, Ala, Leu, and Lys in the human protein. The mutation E1089D showed the same phenotype as MRP1, while the E1089Q substitution markedly decreased resistance to anthracyclines without affecting LTC(4) and E(2)17betaG transport. Conversion of Glu-1089 to Asn, Ala, or Leu had a similar effect on resistance to anthracyclines, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC(4), E(2)17betaG, and the GSH-dependent substrate, estrone-3-sulfate. These results demonstrate that an acidic amino acid residue at position 1089 in predicted TM14 of MRP1 is critical for the ability of the protein to confer drug resistance particularly to the anthracyclines, but is not essential for its ability to transport conjugated organic anions such as LTC(4) and E(2)17betaG.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding Sites; Biological Transport; Cell Line; Cell Membrane; Cell Survival; Doxorubicin; Drug Resistance, Multiple; Estradiol; Estrone; Etoposide; Humans; Kinetics; Leukotriene C4; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Secondary; Recombinant Proteins; Transfection; Vincristine

We identified three novel transporters structurally belonging to the organic anion transporting polypeptide (OATP) family in humans. Since previously known rat oatp1 to 3 do not necessarily correspond to the human OATPs in terms of either tissue distribution or function, here we designate the newly identified human OATPs as OATP-B, -D and -E, and we rename the previously known human OATP as OATP-A. OATP-C proved to be identical with the recently reported LST1/OATP-2. Expression profiles of the five OATPs and the prostaglandin transporter PGT (a member of OATP family) in human tissues showed that OATP-C is exclusively localized in liver, OATP-A and PGT are expressed in restricted ranges of tissues, and OATP-B, -D and -E show broad expression profiles. OATP-B, -C, -D and -E exhibited transport activity for [(3)H]estrone-3-sulfate as a common substrate. OATP-C has a high transport activity with broad substrate specificity.

Topics: Amino Acid Sequence; Anion Transport Proteins; Antiporters; Biological Transport; Carrier Proteins; Cell Line; Cloning, Molecular; Dinoprostone; DNA-Binding Proteins; Estradiol; Estrone; Gene Expression Profiling; Humans; Molecular Sequence Data; Multigene Family; Organ Specificity; Organic Anion Transporters; Penicillin G; Phylogeny; Physical Chromosome Mapping; Polymorphism, Single Nucleotide; RNA, Messenger; Sequence Alignment; Substrate Specificity; Transfection

Organic anion transporting polypeptide (oatp1) has been cloned from rat liver as one of the transporters responsible for the hepatic uptake of ligands, and its substrate specificity has been determined. However, the contribution of oatp1 to the Na+-independent uptake of ligands into rat hepatocytes remains to be investigated. In the present study, we determined the contribution of oatp1 and examined the uptake of ligands into primary cultured hepatocytes (cultured for 4 h) and into COS-7 cells transiently expressing oatp1 and normalized using estradiol-17beta-D-glucuronide as a reference compound. Western blot analysis indicated that oatp1 was less extensively glycosylated in transfected COS-7 cells, and the expression level in transfectant was one-seventh that in rat liver. The Km values for the uptake of estradiol-17beta-D-glucuronide were similar for cultured hepatocytes and oatp1-transfected COS-7 cells (Km = 12.3 versus 20.4 microM), although the Vmax value for oatp1-transfected COS-7 cells was one-seventh that for cultured hepatocytes (Vmax = 1.30 versus 0.175 nmol/min/mg protein). The contribution of oatp1 to the Na+-independent uptake of taurocholic acid and cholic acid into rat hepatocytes was more than 50 to 60%, whereas the corresponding values for the sulfate-conjugates of estrone and 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole were 20 to 30%. In addition, the analysis indicated that the contribution of oatp1 to the Na+-independent uptake of several ligands [glucuronide-conjugate of 6-hydroxy-5, 7-dimethyl2-methylamino-4-(3-pyridylmethyl)benzothiazole, ibuprofen, pravastatin, ouabain, and 2,4-dinitrophenyl-S-glutathione] was minimal. Collectively, the transfected COS-7 cells may be used to quantitatively predict oatp1 activity in hepatocytes after correction of its expressed amount. It is also suggested that multiple transport mechanisms are responsible for the Na+-independent uptake of organic anions into hepatocytes.

Topics: Animals; Anion Transport Proteins; Biological Transport; Blotting, Northern; Blotting, Western; Carrier Proteins; Cells, Cultured; COS Cells; Estradiol; Kinetics; Ligands; Liver; Male; Rats; Rats, Sprague-Dawley; Sodium; Substrate Specificity; Transfection

The rat liver organic anion transporting polypeptide (Oatp1) has been extensively characterized mainly in the Xenopus laevis expression system as a polyspecific carrier transporting organic anions (bile salts), neutral compounds, and even organic cations. In this study, we extended this characterization using a mammalian expression system and confirm the basolateral hepatic expression of Oatp1 with a new antibody. Besides sulfobromophthalein [Michaelis-Menten constant (Km) of approximately 3 microM], taurocholate (Km of approximately 32 microM), and estradiol- 17beta-glucuronide (Km of approximately 4 microM), substrates previously shown to be transported by Oatp1 in transfected HeLa cells, we determined the kinetic parameters for cholate (Km of approximately 54 microM), glycocholate (Km of approximately 54 microM), estrone-3-sulfate (Km of approximately 11 microM), CRC-220 (Km of approximately 57 microM), ouabain (Km of approximately 3,000 microM), and ochratoxin A (Km of approximately 29 microM) in stably transfected Chinese hamster ovary (CHO) cells. In addition, three new substrates, taurochenodeoxycholate (Km of approximately 7 microM), tauroursodeoxycholate (Km of approximately 13 microM), and dehydroepiandrosterone sulfate (Km of approximately 5 microM), were also investigated. The results establish the polyspecific nature of Oatp1 in a mammalian expression system and definitely identify conjugated dihydroxy bile salts and steroid conjugates as high-affinity endogenous substrates of Oatp1.

Topics: Animals; Anion Transport Proteins; Carrier Proteins; CHO Cells; Cholic Acid; Cricetinae; Dehydroepiandrosterone Sulfate; Dipeptides; Estradiol; Estrone; Glycocholic Acid; HeLa Cells; Humans; Kinetics; Liver; Ochratoxins; Ouabain; Piperidines; Rats; Recombinant Proteins; Substrate Specificity; Sulfobromophthalein; Taurochenodeoxycholic Acid; Taurocholic Acid; Transfection; Xenopus laevis

One minute after instillation of 14C-estradiol-17 beta (14C-E2 17 beta) into selected sections of the gastrointestinal tract of swine, radioactive estradiol metabolites were present in blood collected from the portal and jugular veins. Ether was used to extract free but not conjugated estrogens. The percentage of plasma radioactivity that was ether extractable (EE) was low in portal plasma and even lower in jugular plasma following instillation of 14C-E2 17 beta into the stomach, ileum and colon. EE radioactivity was not detectable in either portal or jugular plasma when estradiol was instilled into the duodenum or jejunum. Therefore, estrogens were conjugated either in the lumen of the gastrointestinal tract or as they crossed the intestinal mucosa. The liver played only a minor role in conjugation of these steroids, since the estrogen metabolites present in portal plasma were very similar to those in jugular plasma, and metabolites in the urine were similar to those in plasma. The principal estrogen conjugate found in both portal and jugular plasma, regardless of the gastrointestinal section into which 14C-E2 17 beta was instilled, was estrone glucuronide. There was no uniform metabolic pattern observed in the metabolites of estradiol that remained in the lumen of each gastrointestinal section; however, many metabolic transformations occurred. We concluded that almost all estrogens absorbed were metabolized during the absorption process. The liver was active only in the metabolism of estrogens that escaped conjugation in the intestinal mucosa.

Topics: Animals; Digestive System; Estradiol; Estrone; Female; Intestinal Absorption; Jugular Veins; Liver; Portal Vein; Swine

Topics: Adult; Estradiol; Estriol; Estrogens, Conjugated (USP); Estrone; Humans; Male; Middle Aged