A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane

We cloned and expressed a new organic anion transporting polypeptide (OATP), termed human OATP2, (OATP-C, LST-1; symbol SLC21A6), involved in the uptake of various lipophilic anions into human liver. The cDNA encoding OATP2 comprised 2073 base pairs, corresponding to a protein of 691 amino acids, which were 44% identical to the known human OATP. An antibody directed against the carboxy terminus localized OATP2 to the basolateral membrane of human hepatocytes. Northern blot analysis indicated a strong expression of OATP2 only in human liver. Transport mediated by recombinant OATP2 and its localization were studied in stably transfected Madin-Darby canine kidney strain II (MDCKII) and HEK293 cells. Confocal microscopy localized recombinant OATP2 protein to the lateral membrane of MDCKII cells. Substrates included 17beta-glucuronosyl estradiol, monoglucuronosyl bilirubin, dehydroepiandrosterone sulfate, and cholyltaurine. 17beta-Glucuronosyl estradiol was a preferred substrate, with a Michaelis-Menten constant value of 8.2 microM; its uptake was Na(+) independent and was inhibited by sulfobromophthalein, with a inhibition constant value of 44 nM. Our results indicate that OATP2 is important for the uptake of organic anions, including bilirubin conjugates and sulfobromophthalein, in human liver.     

Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide

Based on sequence homology to the human organic anion transporting polypeptide 2 (OATP2; SLC21A6), we cloned a new member of the SLC21A superfamily of solute carriers, termed OATP8 (SLC21A8). The protein of 702 amino acids showed an amino acid identity of 80% with human OATP2. Based on Northern blotting, the expression of OATP8 was restricted to human liver. Cosmid clones containing the genes encoding human OATP1 (SLC21A3), OATP2 (SLC21A6), and OATP8 (SLC21A8) served to establish their genomic organization. All three genes contained 14 exons with 13 identical splice sites when transferred to the amino acid sequence. An antibody raised against the carboxyl terminus localized OATP8 to the basolateral membrane of human hepatocytes and the recombinant glycoprotein, expressed in MDCKII cells, to the lateral membrane. Transport properties of OATP8 were studied in stably transfected MDCKII and HEK293 cells. Organic anions transported by human OATP8 included sulfobromophthalein, with a K(m) of 3.3 microm, and 17beta-glucuronosyl estradiol, with a K(m) of 5.4 microm. Several bile salts were not substrates. Thus, human OATP8 is a new uptake transporter in the basolateral hepatocyte membrane with an overlapping but distinct substrate specificity as compared with OATP2, which is localized to the same membrane domain.     

The human organic anion transport protein SLC21A6 is not sufficient for bilirubin transport

A recent study (Cui, Y., Konig, J., Leier, I., Buchholz, U., and Keppler, D. (2001) J. Biol. Chem. 276, 9626-9630) suggests that human OATP2 (SLC21A6), also known as OATP-C and LST1, mediates hepatic bilirubin transport. Because of methodologic concerns, this study was designed to examine this issue using a bilirubin transport assay that was validated in overnight cultured rat hepatocytes. These studies showed that cultured rat hepatocytes transported bilirubin with kinetics virtually identical to the transport of sulfobromophthalein. This assay was then used to quantify bilirubin transport by HeLa cells that had been stably transfected with OATP2 under regulation of a metallothionein promoter. Immunoblot analysis revealed abundant expression of OATP2 after incubation of cells for 48 h in zinc, whereas uninduced cells had no expression of this protein. In OATP2-expressing (zinc-induced) HeLa cells at 37 degrees C, the uptake of [35S]sulfobromophthalein was substantial (51.6 +/- 16.5 pmol/15 min/mg protein, n = 5) with little cell-associated ligand in non-expressing (uninduced) cells (0.54 +/- 0.16 pmol/15 min/mg protein, n = 5, p < 0.002). In contrast, there was no difference (p > 0.2) in cell-associated [3H]bilirubin in induced (OATP2-expressing) as compared with uninduced cells (11.25 +/- 3.02 pmol/15 min/mg protein versus 9.15 +/- 1.68 pmol/min/mg protein, respectively, n = 5) We obtained similar results in OATP2-transfected HEK293 cells that were used in the original report. The existence of a bilirubin transporter has been an important field of investigation for many years. Although the current study indicates that a role for OATP2 in hepatocyte bilirubin transport is unlikely, it provides new and sensitive tools that can be adapted to examine the function of putative bilirubin transporters in the future.     

Effect of cryopreservation on the activity of OATP1B1/3 and OCT1 in isolated human hepatocytes

Drug metabolism in liver is the major pathway for xenobiotic elimination from the body. Access to intracellular metabolising enzymes is possible through passive diffusion of lipophilic drugs through cell membrane or active uptake of more polar drugs by specific uptake transporters. Organic Anion Transporting Polypeptides (OATP/SLCO) and Organic Cation Transporters (OCT/SLC22A) are among the most important transporters involved in xenobiotic transport into hepatocytes. Isolated hepatocytes are the model of choice for drug metabolism and drug transport investigations. These primary cells are used either as fresh directly after isolation from liver biopsies, or after subsequent cryopreservation in liquid nitrogen. While cryopreserved hepatocytes are a more convenient and flexible tool for in vitro investigations, information on the functionality of transporter activity after cryopreservation is still sparse. The present study investigated the effect of cryopreservation of human hepatocytes on the uptake of [(3)H]-estradiol-17β-glucuronide (E(2)17βG, substrate of OATP1B1/3/SLCO1B1/3) and [(3)H]-1-methyl-4-phenylpyridinium (MPP+, substrate of OCT1/SLC22A1) into hepatocytes from 6 and 5 human donors, respectively. The results showed that cryopreserved human hepatocytes display carrier-mediated uptake of E(2)17βG and MPP+. While the affinity of E(2)17βG for OATP1B1/3/SLCO1B1/3 was not affected by cryopreservation (Km unchanged, the Wilcoxon signed pair t test gave p=1), V(max) and CL(uptake) values decreased in average by 47% (p=0.06). The passive diffusion of E(2)17βG decreased significantly after cryopreservation (p=0.03). Cryopreservation did not affect Km, V(max) or the passive diffusion of MPP+ in human hepatocytes. In conclusion, the present study showed that cryopreserved human hepatocytes are useful tool to investigate hepatic uptake mediated by OATP1B1/3/SLCO1B1/3 or OCT1/SLC22A1, two of the most important hepatic uptake transporters.     

Identification of phalloidin uptake systems of rat and human liver

To determine whether the liver toxin phalloidin is transported into hepatocytes by one of the known bile salt transporters, we expressed the sodium-dependent Na+/taurocholate cotransporting polypeptide (Ntcp) and several sodium-independent bile salt transporters of the organic anion transporting polypeptide (OATP/SLCO) superfamily in Xenopus laevis oocytes and measured uptake of the radiolabeled phalloidin derivative [3H]demethylphalloin. We found that rat Oatp1b2 (previously called Oatp4 (Slc21a10)) as well as human OATP1B1 (previously called OATP-C (SLC21A6)) and OATP1B3 (previously called OATP8 (SLC21A8)) mediate uptake of [3H]demethylphalloin when expressed in X. laevis oocytes. Transport of increasing [3H]demethylphalloin concentrations was saturable with apparent Km values of 5.7 microM (Oatp1b2), 17 microM (OATP1B1) and 7.5 microM (OATP1B3). All other tested Oatps/OATPs as well as the rat liver Ntcp did not transport [3H]demethylphalloin. Therefore, we conclude that rat Oatp1b2 as well as human OATP1B1 and OATP1B3 are responsible for phalloidin uptake into rat and human hepatocytes.     

Identification of phalloidin uptake systems of rat and human liver

To determine whether the liver toxin phalloidin is transported into hepatocytes by one of the known bile salt transporters, we expressed the sodium-dependent Na⁺/taurocholate cotransporting polypeptide (Ntcp) and several sodium-independent bile salt transporters of the organic anion transporting polypeptide (OATP/SLCO) superfamily in Xenopus laevis oocytes and measured uptake of the radiolabeled phalloidin derivative [³H]demethylphalloin. We found that rat Oatp1b2 (previously called Oatp4 (Slc21a10)) as well as human OATP1B1 (previously called OATP-C (SLC21A6)) and OATP1B3 (previously called OATP8 (SLC21A8)) mediate uptake of [³H]demethylphalloin when expressed in X. laevis oocytes. Transport of increasing [³H]demethylphalloin concentrations was saturable with apparent K_m values of 5.7 μM (Oatp1b2), 17 μM (OATP1B1) and 7.5 μM (OATP1B3). All other tested Oatps/OATPs as well as the rat liver Ntcp did not transport [³H]demethylphalloin. Therefore, we conclude that rat Oatp1b2 as well as human OATP1B1 and OATP1B3 are responsible for phalloidin uptake into rat and human hepatocytes.     

Uptake of [(3)H]bilirubin in freshly isolated rat hepatocytes: role of free bilirubin concentration

Hepatocytic transport of physiological concentrations of unconjugated bilirubin (UCB) has not been determined in isolated liver cells. Initial uptake of highly purified [(3)H]UCB was measured in rat hepatocytes in the presence of human serum albumin at various free, unbound UCB concentrations, [UCB]. At [UCB]=42 nM (below aqueous solubility of 70 nM), uptake was strictly temperature dependent; this was much less evident at [UCB]=166 nM (supersaturated). At low, physiological UCB concentrations, specific UCB uptake showed saturative kinetics with an apparent K(m) of 41 nM, indicating carrier-mediated transport. With aqueous supersaturation, UCB entered hepatocytes mainly by passive diffusion.     

Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia

OATP1B1 (a.k.a. OATP-C, OATP2, LST-1, or SLC21A6) is a liver-specific organic anion uptake transporter and has been shown to be a higher affinity bilirubin uptake transporter than OATP1B3. Using human embryonic kidney (HEK 293) cells stably transfected with OATP1B1, we have studied the effects of indinavir, saquinavir, cyclosporin A, and rifamycin SV on human OATP1B1 transport function. These drugs are potent inhibitors of OATP1B1 transport activity in vitro. We further provide evidence that the calculated fraction of OATP1B1 inhibited at the clinical exposure level correlated very well with the observed hyperbilirubinemia outcome for these drugs in humans. Our data support the hypothesis that inhibition of OATP1B1 is an important mechanism for drug-induced unconjugated hyperbilirubinemia. Inhibition of OATPs may be an important mechanism in drug-drug and drug-endogenous substance interactions.     

Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats

Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.     

Identification of a new urate and high affinity nicotinate transporter, hOAT10 (SLC22A13)

The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine. hORCTL3-expressing Xenopus laevis oocytes showed uptake of [(3)H]nicotinate, [(3)H]p-aminohippurate, and [(14)C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [(3)H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (K(m)) of 22 and 44 microm, respectively, suggesting that hOAT10 may be the molecular equivalent of the postulated high affinity nicotinate transporter in kidneys and intestine. The pH dependence of hOAT10 suggests p-aminohippurate(-)/OH(-), urate(-)/OH(-), and nicotinate(-)/OH(-) exchange as possible transport modes. Urate inhibited [(3)H]nicotinate transport by hOAT10 with an IC(50) value of 759 microm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [(14)C]urate uptake was elevated by an exchange with l -lactate, pyrazinoate, and nicotinate. Surprisingly, we have detected urate(-)/glutathione exchange by hOAT10, consistent with an involvement of hOAT10 in the renal glutathione cycle. Uricosurics, diuretics, and cyclosporine A showed substantial interactions with hOAT10, of which cyclosporine A enhanced [(14)C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.     

Hepatic uptake of bilirubin diglucuronide: analysis by using sinusoidal plasma membrane vesicles

In order to characterize the mechanism for bilirubin transport in the liver, the uptake of bilirubin diglucuronide (BDG) into purified sinusoidal plasma membrane vesicles was investigated. BDG uptake was saturable, and was inhibited by sulfobromophthalein and unconjugated bilirubin, but was not affected by sodium taurocholate. BDG uptake was sodium-independent and was stimulated by intravesicular bilirubin or BDG (trans-stimulation). BDG transport showed strong potential sensitivity; vesicle inside-negative membrane potential created by different anion gradients inhibited BDG uptake whereas vesicle inside-positive membrane potential generated by potassium gradients and valinomycin markedly stimulated BDG transport. These data suggest that BDG, sulfobromophthalein, and probably unconjugated bilirubin share a common transporter in liver cells which is sodium independent, membrane-potential-dependent and capable of exchange. The direction of transport in vivo may be governed by the intracellular concentration of BDG and of other yet unidentified organic anions sharing this transporter.     

Metabolism of dehydroepiandrosterone sulfate and estrone-sulfate by human platelets

The aim of the present research was to study the uptake of DHEAS, and to establish the intracrine capacity of human platelets to produce sex steroid hormones. The DHEAS transport was evaluated through the uptake of [(3)H]-DHEAS in the presence or absence of different substrates through the organic anion transporting polypeptide (OATP) family. The activity of sulfatase enzyme was evaluated, and the metabolism of DHEAS was measured by the conversion of [(3)H]-DHEAS to [(3)H]-androstenedione, [(3)H]-testosterone, [(3)H]-estrone and [(3)H]-17beta-estradiol. Results indicated the existence in the plasma membrane of an OATP with high affinity for DHEAS and estrone sulphate (E(1)S). The platelets showed the capacity to convert DHEAS to active DHEA by the steroid-sulfatase activity. The cells resulted to be a potential site for androgens production, since they have the capacity to produce androstenedione and testosterone; in addition, they reduced [(3)H]-estrone to [(3)H]-17beta-estradiol. This is the first demonstration that human platelets are able to import DHEAS and E(1)S using the OATP family and to convert DHEAS to active DHEA, and to transform E(1)S to 17beta-estradiol.     

SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

Characterization and identification of steroid sulfate transporters of human placenta

Human trophoblasts depend on the supply of external precursors, such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16 alpha-OH-DHEA-S, for synthesis of estrogens. The aim of the present study was to characterize the uptake of DHEA-S by isolated mononucleated trophoblasts (MT) and to identify the involved transporter polypeptides. The kinetic analysis of DHEA-(35)S uptake by MT revealed a saturable uptake mechanism (K(m) = 26 microM, V(max) = 428 pmol x mg protein(-1) x min(-1)), which was superimposed by a nonsaturable uptake mechanism (diffusion constant = 1.2 microl x mg protein(-1) x min(-1)). Uptake of [(3)H]DHEA-S by MT was Na(+) dependent and inhibited by sulfobromophthalein (BSP), steroid sulfates, and probenecid, but not by steroid glucuronides, unconjugated steroids, conjugated bile acids, ouabain, p-aminohippurate (PAH), and bumetanide. MT took up [(35)S]BSP, [(3)H]estrone-sulfate, but not (3)H-labeled ouabain, estradiol-17beta-glucuronide, taurocholate, and PAH. RT-PCR revealed that the organic anion-transporting polypeptides OATP-B, -D, -E, and the organic anion transporter OAT-4 are highly expressed, and that OATP-A, -C, -8, OAT-3, and Na(+)-taurocholate cotransporting polypeptide (NTCP) are not or are only lowly expressed in term placental tissue and freshly isolated and cultured trophoblasts. Immunohistochemistry of first- and third-trimester placenta detected OAT-4 on cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast. Our results indicate that uptake of steroid sulfates by isolated MT is mediated by OATP-B and OAT-4 and suggest a physiological role of both carrier proteins in placental uptake of fetal-derived steroid sulfates.     

Mechanisms for the transport of unconjugated bilirubin in human trophoblastic BeWo cells

To evaluate mechanisms that mediate passage of unconjugated bilirubin (UCB) across placenta, the transport of [3H]UCB was studied in the human trophoblastic, BeWo cell line. When plotted against the unbound UCB concentration [Bf], uptake exhibited saturative kinetics with a similar apparent Km ( approximately 30 nM) for BeWo cells grown either in polarized (Transwell) or non-polarized fashion (dish). UCB release from cells, but not uptake, was inhibited by sulfobromophthalein but not by taurocholate, and almost abolished by MK571, a specific inhibitor of the activity of multidrug resistance-associated proteins (MRPs). MRP1 and MRP5 were both present in BeWo cells and the expression of MRP1, but not MRP5, was markedly higher in polarized cells. These data indicate that UCB is taken up from the fetal circulation by a still undefined, saturative process not shared by other organic anions and is then excreted to maternal circulation by proteins of the MRP family.     

Role of apolipoprotein D in the transport of bilirubin in plasma

Apolipoprotein D (apo D) is a 30-kDa glycoprotein of unknown function that is associated with high-density lipoproteins (HDL). Because unconjugated bilirubin has been shown to bind apo D with a 0. 8:1 stoichiometry, we examined the contribution of this protein to transport of bilirubin in human plasma. Density gradient centrifugation analysis using physiological concentrations of [(14)C]bilirubin reveals that 9% of unconjugated bilirubin is associated with HDL, with the remaining pigment bound primarily to serum proteins (i.e., albumin). The percentage of total plasma bilirubin bound to HDL was found to increase proportionally with bilirubin concentration. Affinity of human apo D for bilirubin was determined by steady-state fluorescence quenching, with Scatchard analysis demonstrating a single binding site for unconjugated bilirubin with an affinity constant (K(a)) of approximately 3 x 10(7) M(-1). Incorporation of apo D into phosphatidylcholine vesicles had no effect on K(a), suggesting that a lipid environment does not alter the affinity of the protein for bilirubin. Using stopped-flow techniques, the first-order rate constant for bilirubin dissociation from apo D was measured at 5.4 s(-1) (half-time = 129 ms). Our findings indicate that HDL is the principal nonalbumin carrier of bilirubin in human plasma and further support the proposition that the affinity of HDL for bilirubin is primarily the result of binding to apo D.     

Novel cationic and neutral glycocholic acid and polyamine conjugates able to inhibit transporters involved in hepatic and intestinal bile acid uptake

To obtain novel drugs able to inhibit transporters involved in bile acid uptake, three compounds were synthesized by conjugating N-(3-aminopropyl)-1,3-propanediamine (PA) with one (BAPA-3), two (BAPA-6), or three (BAPA-8) moieties of glycocholic acid (GC) through their carboxylic group. The expected net charge in aqueous solutions was 2+ (BAPA-3), 1+ (BAPA-6), and 0 (BAPA-8). They were purified by liquid chromatography and their purity checked by HPLC before being chemically characterized by elemental analysis, NMR, and FAB-MS. Using brush-border membranes isolated from rat ileum; their ability to inhibit [(14)C]-GC transport (BAPA-3>BAPA-6>BAPA-8) was suggested. This was further investigated 48h after injecting Xenopus laevis oocytes with the mRNA of rat sodium/taurocholate (TC)-cotransporting polypeptide (Ntcp), rat apical sodium-dependent bile salt transporter (Asbt), or the human isoforms OATP-C/1B1 and OATP8/1B3 of organic anion-transporting polypeptides, when maximal functional expression was detected. BAPA-8, BAPA-6, and BAPA-3 induced no inhibition of OATP8/1B3-mediated [(3)H]-TC uptake, but dramatically reduced [(3)H]-TC uptake by OATP-C/1B1. In the cases of Ntcp- and Asbt-mediated [(3)H]-TC uptake, these were sodium-dependent and were inhibited by BAPA-6>BAPA-8>BAPA-3 and BAPA-8>BAPA-6>BAPA-3, respectively. In conclusion, our results suggest that these compounds are potentially interesting research tools for the selective modulation of liver and intestinal uptake of bile acids and other cholephilic compounds. Moreover, they may be of pharmacological usefulness to prevent the acute toxicity of compounds reaching liver cells through specific transporters or to enhance both fecal elimination of bile acids and hence cholesterol consumption for the 'de novo' synthesis of bile acids.     

Transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and Multidrug resistance-associated protein 2 (MRP2/ABCC2).

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.     

Preparation of [35S]sulfobromophthalein of high specific activity

Study of the hepatocyte transport mechanism of organic anions such as bilirubin and sulfobromophthalein has been limited by the relatively low specific activities of these ligands. [3H]Bilirubin and [35S]sulfobromophthalein have been available with specific activities of only approximately 100 mCi/mmol. We now report a relatively simple procedure to prepare [35S]sulfobromophthalein at a specific activity of approximately 3000 mCi/mmol. This compound is radiochemically pure and serves as a tracer for authentic sulfobromophthalein as judged by chromatography, hepatocyte uptake, metabolism, and biliary excretion. Use of this material as a photoaffinity probe and as a transported ligand may permit dissection and understanding of its transport mechanism.