Heterogeneous accumulation of fluorescent bile acids in primary rat hepatocytes does not correlate with their homogenous expression of ntcp

Sodium taurocholate-cotransporting polypeptide (ntcp) is considered to be a major determinant of bile acid uptake into hepatocytes. However, the regulation of ntcp and the degree that it participates in the accumulation of specific substrates are not well understood. We utilized fluorescent bile acid derivatives and direct quantitation of fluorescent microscopy images to examine the regulation of ntcp and its role in the cell-to-cell variability of fluorescent bile acid accumulation. Primary-cultured rat hepatocytes rapidly accumulated the fluorescent bile acids, chenodeoxycholylglycylamidofluorescein (CDCGamF), 7-β- nitrobenzoxadiazole 3-α hydroxy 5-β cholan-24-oic acid (NBD-CA), and cholyl-glycylamido-fluorescein (CGamF). However, in stably transfected HeLa cells, ntcp preferred CDCGamF, whereas the organic anion transporter, organic anion transporting polypeptide 1 (oatp1a1), preferred NBD-CA, and neither ntcp nor oatp1a1 showed strong accumulation of CGamF by these methods. Ntcp-mediated transport of CDCGamF was inhibited by taurocholate, cyclosporin, actin depolymerization, and an inhibitor of atypical PKC-ζ. The latter two agents altered the cellular distribution of ntcp as visualized in ntcp-green fluorescent protein-transfected cells. Although fluorescent bile acid accumulation was reproducible by the imaging assays, individual cells showed variable accumulation that was not attributable to changes in membrane permeability or cell viability. In HeLa cells, this was accounted for by variable levels of ntcp, whereas, in hepatocytes, ntcp expression was uniform, and low accumulation was seen in a large portion of cells despite the presence of ntcp. These studies indicate that single-cell imaging can provide insight into previously unrecognized details of anion transport in the complex environment of polarized hepatocytes.     

Expression, transport properties, and chromosomal location of organic anion transporter subtype 3

The rat and mouse organic anion-transporting polypeptides (oatp) subtype 3 (oatp3) were cloned to further define components of the intestinal bile acid transport system. In transfected COS cells, oatp3 mediated Na(+)-independent, DIDS-inhibited taurocholate uptake (Michaelis-Menten constant approximately 30 microM). The oatp3-mediated uptake rates and affinities were highest for glycine-conjugated dihydroxy bile acids. In stably transfected, polarized Madin-Darby canine kidney (MDCK) cells, oatp3 mediated only apical uptake of taurocholate. RT-PCR analysis revealed that rat oatp3, but not oatp1 or oatp2, was expressed in small intestine. By RNase protection assay, oatp3 mRNA was readily detected down the length of the small intestine as well as in brain, lung, and retina. An antibody directed to the carboxy terminus localized oatp3 to the apical brush-border membrane of rat jejunal enterocytes. The mouse oatp3 gene was localized to a region of mouse chromosome 6. This region is syntenic with human chromosome 12p12, where the human OATP-A gene was mapped, suggesting that rodent oatp3 is orthologous to the human OATP-A. These transport and expression properties suggest that rat oatp3 mediates the anion exchange-driven absorption of bile acids previously described for the proximal small intestine.     

Molecular regulation of sinusoidal liver bile acid transporters during cholestasis.

Impairment of the hepatic transport of bile acids and other organic anions will result in the clinically important syndrome of cholestasis. Cloning of a number of specific hepatic organic anion transporters has enabled studies of their molecular regulation during cholestasis. The best characterized transport system is a 50-51 kDa sodium-dependent taurocholate cotransporting polypeptide (ntcp), which mediates the sodium-dependent uptake of conjugated bile acids at the sinusoidal plasma membrane of hepatocytes. Under physiologic conditions and after depletion of biliary constituents, ntcp remains constitutively expressed throughout the liver acinus. However, both function and expression of ntcp are rapidly down-regulated in rat liver in various models of experimental cholestasis, such as cholestasis induced by common bile duct ligation, estrogen, endotoxin or cytokine treatment. In addition to ntcp, the sinusoidal organic anion transporting polypeptide oatp-1 is also down-regulated at the protein and steady-state mRNA levels in estrogen-cholestasis, but does not affect sodium-independent uptake of taurocholate. The regulation of a recently cloned member of the organic anion transporter family (oatp-2), which is highly expressed in liver, remains to be studied under cholestatic conditions.     

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.     

Hepatocellular transport of cyclosomatostatins: evidence for a carrier system related to the multispecific bile acid transporter.

The uptake of the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, was studied in isolated hepatocytes. 008 is taken up by hepatocytes in a concentration-, time-, energy- and temperature- dependent manner. Since 008 is hydrophobic, it binds rapidly to liver cells. This is evident by the positive intercept at the gamma-axis in the uptake curves. At higher concentrations, a minor part of the transport occurs by diffusion at a rate of 8.307.10(-6) cm/s. This part of diffusion is measured at 4 degrees C and can be subtracted from the uptake at 37 degrees C resulting in the carrier mediated part of uptake which is saturable. Kinetic parameters for the saturable part of uptake are Km 1.5 microM and Vmax 40.0 pmol/mg per min. The transport is decreased in the absence of oxygen and in the presence of metabolic inhibitors. Uptake is accelerated at temperatures above 20 degrees C. The activation energy was determined to be 30.77 kJ/mol. The membrane potential and not a sodium gradient is the main driving force for 008 transport. Cholate (a typical substrate of the multispecific bile acid transporter) and taurocholate are mutual competitive inhibitors of 008 uptake. Phalloidin, antamanide and iodipamide, typical foreign substrates of the transporter, interfere with the uptake of 008. AS 30D ascites hepatoma cells, known to be unable to transport bile acids, phalloidin and iodipamide, are also unfit to transport 008. Interestingly, sulfobromophthalein (BSP) but not rifampicin, both foreign substrates of the bilirubin carrier, inhibits the transport of 008 in a competitive manner.     

Characteristics of sinusoidal uptake and biliary excretion of cysteinyl leukotrienes in perfused rat liver

In single-pass perfused rat liver, the sinusoidal uptake of infused 3H-labelled leukotriene (LT) C4 (10 nmol.l-1) was inhibited by sulfobromophthalein. Inhibition was half-maximal at sulfobromophthalein concentrations of approximately 1.2 mumol.l-1 in the influent perfusate and leukotriene uptake was inhibited by maximally 34%. Sulfobromophthalein (20 mumol.l-1) also decreased the uptake of infused [3H]LTE4 (10 nmol.l-1) by 31%. Indocyanine green (10 mumol.l-1) inhibited the sinusoidal [3H]LTC4 uptake by 19%. Replacement of sodium in the perfusion medium by choline decreased the uptake of infused [3H]LTC4 (10 nmol.l-1) by 56%, but was without effect on the uptake of sulfobromophthalein. The canalicular excretion of LTC4, LTD4 and N-acetyl-LTE4 was inhibited by sulfobromophthalein. In contrast, the proportion of polar omega-oxidation metabolites recovered in bile following the infusion of [3H]LTC4 was increased. Taurocholate, which had no effect on the sinusoidal leukotriene uptake, increased bile flow and also the biliary elimination of the radioactivity taken up. With increasing taurocholate additions, the amount of LTD4 recovered in bile increased at the expense of LTC4. Following the infusion of [3H]LTD4 (10 nmol.l-1), a major biliary metabolite was LTC4 indicating a reconversion of LTD4 to LTC4. In the presence of taurocholate (40 mumol.l-1), however, this reconversion was completely inhibited. The findings suggest the involvement of different transport systems in the sinusoidal uptake of cysteinyl leukotrienes. LTC4 uptake is not affected by bile acids and has a sodium-dependent and a sodium-independent component, the latter probably being shared with organic dyes. Sulfobromophthalein also interferes with the canalicular transport of LTC4, LTD4 and N-acetyl-LTE4, but not with the excretion of omega-oxidized cysteinyl leukotrienes. The data may be relevant for the understanding of hepatic leukotriene processing in conditions like hyperbilirubinemia or cholestasis.     

Characterization of cloned mouse Na+/taurocholate cotransporting polypeptide by transient expression in COS-7 cells

The mouse Na+/taurocholate cotransporting polypeptide transiently expressed in COS-7 cells caused sodium-dependent uptake of [3H]taurocholic acid with Km and Vmax values of 18 microM and 102 pmol/mg protein/min, respectively. This Km value is comparable to that for rat NTCP and higher than that for human NTCP. Substrate specificity was evaluated by measuring inhibitory effects of unlabeled bile acids on [3H]taurocholic acid transport.     

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.     

Transport of sulfobromophthalein and taurocholate in the HepG2 cell line in relation to the expression of membrane carrier proteins.

The transport of two different classes of organic anions (cholephilic dyes; the sulfobromophthalein, BSP, and bile acids; taurocholate, TC) was investigated in the HepG2 cell line. At 37 degrees C, BSP uptake was found to be biphasic with an apparent saturative curve in the concentration range between 0-6 microM followed by a linear component up to 18 microM. Kinetic constant determination showed an apparent Km of 26.6 +/- 3.1 microM and a Vmax of 5.64 +/- 0.82 nmol BSP.min-1.mg prot-1. At 4 degrees C, uptake was linear. By subtracting this latter component from the total uptake, a saturable, carrier mediated uptake was found with an apparent Km of 3.6 +/- 1.0 microM BSP and a Vmax of 0.37 +/- 0.04 nmol BSP.min-1.mg prot-1 (m +/- SEM, n = 6). These values were fully comparable with those found in freshly isolated male hepatocyte. Immunoblot analysis of HepG2 cell plasma membrane revealed the presence of bilitranslocase when tested against a monospecific antibody against this carrier molecule. On the contrary, TC uptake was linear up to concentration of 100 microM TC. No difference was observed in the presence or absence of Na+. Immunoprecipitation analysis showed the absence of the putative carrier of TC. These data indicate that the HepG2 cell line expresses a functioning bilitranslocase-mediated system. Conversely, carrier mediated bile acid uptake is absent in line with the lack of expression of the carrier protein.     

Synthesis and transport characteristics of photoaffinity probes for the hepatocyte bile acid transport system

In an effort to characterize the hepatocyte bile acid transport system, a photoreactive derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid (7-ADTC) has been synthesized and its transport properties compared to those of the natural substrate. Both the bile acid and its synthetic analog were shown to be transported against an electrochemical gradient as well as a chemical gradient. Transport as a function of concentration and the presence of sodium indicated that both substrates were taken up by a sodium-dependent and a sodium-independent route. Taurocholate had Km values of 26 and 57 microM and Vmax values of 0.77 and 0.15 nmol/mg of protein/min, respectively. In comparison, 7-ADTC had very similar kinetic properties with Km values of 25 and 31 microM and Vmax values of 1.14 and 0.27 nmol/mg of protein/min. Each compound was shown to inhibit competitively the transport of the other, suggesting that these substrates utilized a common membrane carrier. The transport properties of the photoreactive anion transport inhibitor, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) were also characterized in the hepatocyte system. Transport occurred via a sodium-dependent and a sodium-independent route with Km values of 210 and 555 microM and Vmax values of 0.57 and 1.62 nmol/mg of protein/min. As in the case of 7-ADTC, NAP-taurine and taurocholate were also shown to be mutual competitive inhibitors. In the absence of light, 7-ADTC was a reversible inhibitor of taurocholate uptake. Upon irradiation, irreversible photoinactivation of the taurocholate uptake system was observed. These results indicate that 7-ADTC and NAP-taurine can be utilized as photoaffinity probes for the identification of the bile acid carrier protein(s) in hepatocyte plasma membranes.     

Terminal deoxynucleotidyl transferase in AKR leukemic cells and lack of relation of enzyme activity to cell cycle phase.

Cholate and taurocholate uptakes were studied in presence of albumin using isolated rat hepatocytes. Albumin decreased nonspecific binding of both bile acids and inhibited cholate uptake noncompetitively and taurocholate uptake competitively. Although different bile acids except dehydrocholate inhibited both cholate and taurocholate uptake, their relative inhibitory potency was not the same for both bile acids. Uptake of both bile acids was characterized by a saturable as well as an unsaturable process both in presence and in absence of albumin. The results suggest that both bile acids may be transported by more than one carrier and taurocholate is transported more efficiently than cholate by hepatocytes.     

Two distinct mechanisms for taurocholate uptake in subcellular fractions from rat liver

As part of the enterohepatic circulation, hepatocytes take up bile acids from the intestines via the hepatic portal blood using a sodium-dependent carrier mechanism and resecrete the bile acids into the bile. In order to assess whether intracellular organelles are involved in the transcellular secretion of bile acids, we measured directly the ability of purified subcellular fractions of rat liver to take up taurocholate using a Millipore filtration assay. Two distinct uptake mechanisms can be discerned, one localized in the plasma membranes and the other in the Golgi and smooth microsomal fractions. Plasma membranes prepared by the method of Fleischer and Kervina (Fleischer, S., and Kervina, M. (1974) Methods Enzymol. 31, 6) take up taurocholate in a saturable manner with an apparent Vmax of 2.4 nmol min-1 mg protein-1 and a Km of 190 microM at 37 degrees C. After preincubation of the membranes with K+ ions, a sodium gradient (100 mM outside) stimulates the uptake rate by 90% with the observed Km unchanged. The stimulation is inhibited by phalloidin but not by bromosulfophthalein. Bile canalicular plasma membranes made according to Kramer et al. (Kramer, W., Bickel, U., Buscher, H. P., Gerok, W., and Kurz, G. (1982) Eur. J. Biochem. 129, 13-24) do not take up taurocholate. The transport by Golgi vesicles and smooth microsomes differs from that in the plasma membrane fraction in that it is not stimulated by a sodium gradient, has a Vmax of 12 nmol min-1 mg protein-1 and a Km of 440 microM at 37 degrees C, and is inhibited by bromosulfophthalein but not by phalloidin. Taurocholate uptake into smooth microsomes is abolished by filipin, an antibiotic that complexes with cholesterol to disrupt the membrane. This suggests that taurocholate uptake occurs into a nonendoplasmic reticulum subfraction since endoplasmic reticulum membranes contain negligible amounts of cholesterol. Little uptake was observed using rough microsomes or mitochondria. A model of transhepatic transport compatible with our observations is that taurocholate uptake into the cytoplasm occurs via the plasma membranes on the sinusoidal side of the hepatocyte; taurocholate is then taken up into smooth vesicles and the Golgi complex and is secreted into the bile by exocytosis as the vesicles fuse with the canalicular plasma membranes.     

Down-regulation by extracellular ATP of rat hepatocyte organic anion transport is mediated by serine phosphorylation of oatp1

Recent studies implicate a role in hepatocyte organic anion transport of a plasma membrane protein that has been termed oatp1 (organic anion transport protein 1). Little is known regarding mechanisms by which its transport activity is modulated in vivo. In previous studies (Campbell, C. G., Spray, D. C., and Wolkoff, A. W. (1993) J. Biol. Chem. 268, 15399-15404), we demonstrated that hepatocyte uptake of sulfobromophthalein was down-regulated by extracellular ATP. We have now found that extracellular ATP reduces the V(max) for transport of sulfobromophthalein by rat hepatocytes; K(m) remains unaltered. Reduced transport also results from incubation of hepatocytes with the phosphatase inhibitors okadaic acid and calyculin A. Immunoprecipitation of biotinylated cell surface proteins indicates that oatp1 remains on the cell surface after exposure of cells to ATP or phosphatase inhibitor, suggesting that loss of transport activity is not caused by transporter internalization. Exposure of (32)P-loaded hepatocytes to extracellular ATP results in serine phosphorylation of oatp1 with the appearance of a single major tryptic phosphopeptide; oatp1 from control cells is not phosphorylated. This phosphopeptide comigrates with one of four phosphopeptides resulting from incubation of cells with okadaic acid. These studies indicate that the phosphorylation state of oatp1 must be an important consideration when assessing alterations of its functional expression in pathobiological states.     

Bile secretion in hemoglobin-free perfused rat liver

Hemoglobin-free perfused rat liver was demonstrated to be a suitable experimental model in studying bile secretion. Bile flow slowly decreased to more than 3 h of perfusion. Despite differences in metabolic states, the bile flow was the same in the recirculating as in the nonrecirculating mode of perfusion. Sulfobromophthalein stimulated bile flow at high rates of infusion. In bile, the ratio conjugated to unconjugated sulfobromophthalein also increased with sulfobromophthalein infusion rate. The access of [14C]insulin, [14C] sucrose, and inorganic [32P] phosphate from perfusate into bile was restricted. Bile flow, secretion of taurocholate and sulfobromophthalein, and bile pressure are compared with values from anesthetized animals and from isolated livers perfused with medium containing erythrocytes.     

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.     

Investigations on the sodium dependence of bile acid fluxes in the isolated perfused rat liver.

At [Na+]o = 118 mM the concentrative transfer of cholic and taurocholic acid from the perfusate into the isolated rat liver displays saturation kinetics (taurocholate: V = 299 nmol-min-1-g-1, Km = 61 muM; Cholate: V=327 nmol-min-1-g-1, Km = 436 muM). Perfusion with an isotonic sodium-free medium did not change the feature of a carrier-mediated transport but did markedly reduce V without affecting Km (taurocholate: V = 65 nmol-min-1-g-1, Km = 78 muM; cholate: V = 104 nmol-min-1-g-1, Km = 354 muM). It was experimentally assured that the observed reduction of bile salt uptake was not a consequence of regurgitation of bile salts or due to an excessive intracellular accumulation during cholestasis in the sodium-free state. The rate of taurocholate efflux is very low when compared with the rapid rate of the uptake. A stimulatory action of extracellular sodium on this pathway was also observed. Inhibition of the (Na+ + K+)-ATPase by 1 mM ouabain resulted in a decrease of bile salt uptake. Activation of the enzyme by potassium readmission to a K+-deprived liver enhanced bile salt uptake. The immediate response to alteration of the enzyme activity suggests a close association of a fraction of bile acid active transport with the sodium pump.     

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.     

Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum.

The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.     

Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6

Bilirubin, the end product of heme catabolism, is taken up from the blood circulation into the liver. This work identifies a high-affinity transport protein mediating the uptake of bilirubin and its conjugates into human hepatocytes. Human embryonic kidney cells (HEK293) permanently expressing the recombinant organic anion-transporting polypeptide 2 (human OATP2, also known as LST-1 or OATP-C; symbol SLC21A6) showed uptake of [(3)H]monoglucuronosyl bilirubin, [(3)H]bisglucuronosyl bilirubin, and [(3)H]sulfobromophthalein with K(m) values of 0.10, 0.28, and 0.14 microm, respectively. High-affinity uptake of unconjugated [(3)H]bilirubin by OATP2 occurred in the presence of albumin and was not mediated by another basolateral hepatic uptake transporter, human OATP8 (symbol SLC21A8). OATP2 and OATP8 differed by their capacity to extract substrates from albumin before transport. In comparison to the high-affinity transport by OATP2, OATP8 transported [(3)H]sulfobromophthalein and [(3)H]monoglucuronosyl bilirubin with lower affinity, with K(m) values of 3.3 and 0.5 microm, respectively. The organic anion indocyanine green potently inhibited transport mediated by OATP2, with a K(i) value of 112 nm, but did not inhibit transport mediated by OATP8. Human OATP2 may play a key role in the prevention of hyperbilirubinemia by facilitating the selective entry of unconjugated bilirubin and its glucuronate conjugates into 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 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.