Binding proteins for linear renin-inhibiting peptides in basolateral plasma membranes of rat liver.

A linear hydrophobic peptide, (Code no. EMD 55068), a synthetic renin-antagonist, competitively inhibits the uptake of taurocholate and of another linear peptide (EMD 51921) but not of oleic acid, serine or thiamin hydrochloride into isolated rat liver cells. EMD 55068 was attached to a gel matrix at a position that is not involved in the protein ligand interaction. The gel matrix used did not interact nonspecifically with solubilized proteins from rat liver. The quantity of bound ligand was determined to be 3.6 mg/ml of gel matrix. In the fraction of EDTA extracted hydrophilic membrane-associated proteins, no binding proteins were detected. Affinity chromatography of integral plasma membrane proteins resulted in four protein bands with molecular masses of 46, 49, 53 and 56 kDa in SDS-PAGE. In contrast, solubilized plasma membrane proteins from AS-30D ascites hepatoma cells, which are unable to transport bile acids and linear peptides, did not bind specifically to the affinity matrix.     

New substrates of the multispecific bile acid transporter in liver cells: interference of some linear renin inhibiting peptides with transport protein(s) for bile acids

Interactions between some stable linear peptides with renin inhibitory activity and a multispecific transport system in the basolateral plasma membrane of liver cells was studied on cell suspensions. The peptides used in our experiments were taken up by liver cells and subsequently eliminated without any biotransformation (e.g., proteolysis). No degradation products could be detected in the extracellular medium by thin-layer chromatography. All peptides tested inhibited the uptake of physiological and of some foreign substrates of the multispecific bile acid transporter (MT). The phalloidin response of liver cells was also inhibited to a similar degree in a concentration-dependent manner. The potency of inhibition did not correlate with the lipophilic properties of the peptides. On the other hand a tight correlation could be documented between the inhibition of cholate transport and that of the phalloidin response. Transport inhibition of typical substrates of the MT by the above renin inhibitors was competitive. In contrast, the transport of a typical substrate of the bilirubin carrier (rifampicin), of amino acids (alpha-aminoisobutyric acid), long chain fatty acids (oleic acid) and cationic compounds (thiamin hydrochloride) was not inhibited by the same renin inhibitors. These results indicate that linear renin inhibiting peptides are taken up into liver cells by carrier proteins related to the MT.     

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.     

Hepatocellular uptake of aflatoxin B1 by non-ionic diffusion. Inhibition of bile acid transport by interference with membrane lipids

Aflatoxin B1 permeates isolated rat hepatocytes by non-ionic diffusion. Its uptake is neither saturable nor influenced by metabolic energy and not inhibited by treatment of cells with proteases. The initial rate of aflatoxin B1 uptake measured at 7 degrees C is between 40 and 50% compared to that at 37 degrees C. However, after an incubation period of 7 minutes identical equilibrium uptake is reached at both temperatures. The apparent activation energies, calculated for aflatoxin B1 uptake by Arrhenius diagrams ranged between 1.69 and 4.5 kcal/mol. A Q10 value of 1.34 was calculated for a temperature interval of 7-17 degrees C but decreased to 1.05 for the interval of 27-37 degrees C. Liposomes or lipoproteins added to the cell suspension inhibited the aflatoxin B1 uptake into hepatocytes. Liposomes mainly composed of unsaturated fatty acids bind twice as much aflatoxin B1 as those composed of saturated ones, indicating that the lipophilicity of the mycotoxin is crucial in the determination of its uptake into liver cells. At concentrations above 5 micrograms/ml, aflatoxin B1 inhibited the carrier-mediated uptake of cholic acid and of phalloidin into hepatocytes. This effect was reversible and abolished by washing the cells after preincubation with aflatoxin. In concentrations below 5 micrograms/ml the uptake of phallotoxin and cholic acid was however stimulated by 15-25%. These results indicate, that a carrier-mediated uptake into hepatocytes via the multispecific bile salt transporter is not responsible for the organoselective clearance of aflatoxins by the liver. On the other hand, the cholestatic effect of aflatoxin B1 results at least partially from the inhibition of the multispecific bile acid transport system. This inhibition may arise from affinity of aflatoxins to lipid domains of the cell membrane.     

Inhibition by sporidesmin of hepatocyte bile acid transport.

Exposure of isolated rat hepatocytes (approx. 2 x 10(7)--5 x 10(7) cells/10ml of incubation mixture) to 0.5 mg of the mycotoxin sporidesmin for 30--60 min at 37 degrees C produced loss of plasma-membrane microvilli with some disruption of organelle distribution in the sub-surface region. There was accompanying inhibition of [14C]cholate and [14C]taurocholate transport, but bile acid conjugation was not altered. Inhibition of cholate uptake was maximal after exposure of hepatocytes to sporidesmin for 1 min, and was not reversed by washing cells free of extracellular sporidesmin. N-Ethylmaleimide (0.1 mM) or dithiothreitol (1 mM) partially protected hepatocytes from sporidesmin inhibition of bile acid uptake. Significant protection was not given by other thiols or by zinc sulphate, cholesterol, ascorbate or alpha-tocopherol. The results are discussed in terms of sporidesmin action on cell membranes and the toxin's effect on bile secretion.     

Expression of the hepatocyte Na+/bile acid cotransporter in Xenopus laevis oocytes

The expression of the basolateral Na+/bile acid (taurocholate) cotransport system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of rat liver poly(A)+ RNA into the oocytes resulted in the functional expression of Na+ gradient stimulated taurocholate uptake within 3-5 days. This Na(+)-dependent portion of taurocholate uptake exhibited saturation kinetics (apparent Km approximately 91 microM) and could be inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene. Furthermore, the expressed taurocholate transport activity demonstrated similar substrate inhibition and stimulation by low concentrations of bovine serum albumin as the basolateral Na+/bile acid cotransport system previously characterized in intact liver, isolated hepatocytes, and isolated plasma membrane vesicles. Finally, a 1.5- to 3.0-kilobase size-class of mRNA could be identified that was sufficient to express the basolateral Na+/taurocholate uptake system in oocytes. These results demonstrate that "expression cloning" represents a promising approach to ultimately clone the gene and to further characterize the molecular properties of this important hepatocellular membrane transport system.     

Inhibition of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid oxidation and of bile acid secretion in rat liver by fatty acids

In isolated rat hepatocytes, fatty acids inhibited the side chain oxidation, but not the uptake, of exogenously added 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA). THCA did not inhibit fatty acid oxidation. In liver homogenates, fatty acids inhibited THCA activation to its CoA ester (THC-CoA) and THCA oxidation. THCA did not influence fatty acid activation or oxidation. Comparison of the THC-CoA concentrations present in the incubation mixtures during THCA oxidation, with substrate concentration curves determined for THC-CoA oxidation, indicated that the inhibition of THCA oxidation by fatty acids was at least partly exerted at the activation step. The inhibition of THCA activation by fatty acids was noncompetitive. Palmitoyl-CoA at concentrations found in the incubation mixtures during THCA oxidation in the presence of palmitate inhibited THC-CoA oxidation, but not sufficiently to fully explain the fatty acid-induced inhibition of THCA oxidation. The inhibition of THC-CoA oxidation by palmitoyl-CoA did not seem to be competitive. Acyl-CoA oxidase, the first enzyme of peroxisomal beta-oxidation (which catalyzes the side chain oxidation of THCA), was enhanced 15-fold in liver homogenates from clofibrate-treated rats when palmitoyl-CoA was the substrate, but the oxidase activity remained unaltered when THC-CoA was the substrate. In the perfused liver, oleate, infused after a wash-out period of 60 min, markedly inhibited bile acid secretion. The results 1) suggest that fatty acids inhibit THCA metabolism both at the activation step and at the peroxisomal beta-oxidation sequence and that separate enzymes may be involved in both the activation and peroxisomal beta-oxidation of fatty acids and THCA and 2) raise the question whether fatty acids might (indirectly?) affect overall bile acid synthesis via their inhibitory effect on THCA metabolism.     

Influence of gastrointestinal peptides on bile acid transport by isolated rat hepatocytes

While numerous effects of gut peptides on gastric, pancreatic, and intestinal secretion have been described, there has been little investigation of the influence of these peptides on hepatic function. In the present studies, effects of vasoactive intestinal peptide (VIP), somatostatin, thyrotropin-releasing hormone (TRH), and bombesin on taurocholate transport by isolated rat hepatocytes have been examined. Somatostatin, TRH, and bombesin in incubation media produced no change from control incubations with regard to either uptake of taurocholate by hepatocytes or efflux of bile acid from preloaded cells. However, incubation of hepatocytes with VIP produced a significant decrease in taurocholate uptake (1.34 +/- 0.13 versus 1.73 +/- 0.16 nmole.min-1.10(6) cells-1, P less than 0.001). Studies with verapamil, a calcium-channel blocking agent, and theophylline, an inhibitor of cAMP catabolism, failed to provide evidence for transmembrane Ca2+ flux or alteration in intracellular levels of cAMP, respectively, as mechanisms for the observed inhibition of hepatocyte taurocholate uptake by VIP. These data, coupled with both clinical and other basic observations, suggest that VIP may play a significant role in the regulation of hepatic bile secretion.     

Comparative investigations on the uptake of phallotoxins, bile acids, bovine lactoperoxidase and horseradish peroxidase into rat hepatocytes in suspension and in cell cultures

Two alternative uptake mechanisms for phallotoxins by liver cells are debated: carrier-mediated uptake and receptor-mediated endocytosis. We have compared the properties of hepatocellular uptake of the phallotoxins, phalloidin and demethylphalloin, with the uptake of cholate as a substrate for carrier-mediated uptake and compared with iodinated bovine lactoperoxidase or iodinated horseradish peroxidase, as the latter are known to be taken up by vesicular endocytosis. Uptake of phallotoxins and [14C]cholate uptake into isolated hepatocytes is independent of extracellular calcium but inhibited by A23187 or by monensin. Uptake of bovine lactoperoxidase strictly depends on external Ca2+, was insensitive to A23197 and was not inhibited by monensin. No mutual uptake inhibition between phalloidin or cholate and peroxidases was seen, indicating independent permeation pathways in hepatocytes. However, high concentrations of cytochalasin B inhibited the uptake of either phalloidin, cholate or bovine lactoperoxidase. Horseradish peroxidase uptake, which was taken as an indicator for fluid pinocytosis, was low in isolated hepatocytes and could not account for the amount of phalloidin or cholate taken up. In cultured rat hepatocytes, uptake of phallotoxins decreased within 1 day to 10% of the uptake seen in freshly isolated hepatocytes. The results indicate different mechanisms for hepatocellular phallotoxin/bile-acid uptake and peroxidase internalization. As monolayer cultures of hepatocytes rapidly lost the carrier-mediated uptake of phallotoxins and bile acids, freshly isolated hepatocytes might be a more suitable experimental model than cultured cells for kinetic studies on this transport system.     

Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria

The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.     

Characteristics of taurine transport in rat liver lysosomes

Taurine (2-aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety of cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. Intralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na(+)-independent, with an overshoot between 5-10 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km1 = 31 +/- 1.82 and Km2 greater than 198 +/- 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a t1/2 of 5 minutes at 37 degrees C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane beta-alanine carrier system, with inhibition by beta-alanine, hypotaurine, and taurine. alpha-Alanine, 2-methylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP-taurine [N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate]. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K+ gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of this osmotic regulatory substance.     

Identification of functionally relevant residues of the rat ileal apical sodium-dependent bile acid cotransporter

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na(+) ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na(+) ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na(+).     

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.     

A kinetic study of mitochondrial calcium transport.

This report describes a kinetic analysis of energy-linked Ca2+ transport in rat liver mitochondria, in which a ruthenium red/EGTA [ethanedioxy-bis(ethylamine)-tetraacetic acid] quenching technique has been used to measure rates of 45Ca2+ transport. Accurately known concentrations of free 45Ca2+ were generated with Ca2+/nitrilotriacetic acids buffers for the determination of substrate/velocity relationships. The results show that the initial velocity of transport is a sigmoidal function of Ca2+ concentration (Hill coefficient = 1.7), the Km being 4 muM Ca4 at 0 degrees C and pH 7.4. These values for the Hill coefficient and the Km remain constant in the presence of up to 2 mM phosphate, but with 10 mM acetate both parameters are increased slightly. Both permeant acids increase the maximum velocity to an extent dependent on their concentration. The Ca2+-binding site(s) of the carrier contains a group ionizing at pH approximately 7.5 at 0 degrees C, which is functional in the dissociated state. The stimulatory effect of permeant acids is ascribed to their facilitating the release of Ca2+ from the carrier to the internal phase, an interpretation which is strengthened by the lack of effect of the permeant anion SCN- on Ca2+ transport. Studies on the time-course of Ca2+ uptake and of EFTA-induced Ca2+ efflux from pre-loaded mitochondria demonstrate the reversibility of the carrier in respiring mitochondria and the extent to which this property is influenced by permeant acids. These data are accommodated in a carrier mechanism based on electrophoretic transport of Ca2+ bound to pairs of interacting acidic sites.     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Functional and molecular characterization of a peptide transporter in the rat PC12 neuroendocrine cell line.

We have studied functional properties of peptide transport in the pheochromocytoma neuroendocrine cell line from rat. The neutral peptide D-Phe-L-Ala (resistant to hydrolysis) is a good substrate for uptake into these cells. Transport is substantially inhibited by diethylpyrocarbonate pretreatment and is stimulated by external acidification. It is sodium-independent and, unexpectedly, insensitive to membrane potential. Peptide uptake is inhibited by a wide variety of other di- and tripeptides but not by amino acids. The neuropeptide kyotorphin (opioid dipeptide (L-Tyr-L-Arg)) inhibits uptake of labelled peptide and trans-stimulates efflux showing that it is a transported substrate. These findings are discussed in relation to the molecular basis and physiological role of this transport system.     

Peptide substrates rapidly modulate expression of dipeptide and oligopeptide permeases in Candida albicans

Previous studies showed that peptide transport activity in Candida albicans was completely repressed by NH4+, and that growth on amino acids as sole nitrogen source stimulated transport to a basal level. Here we show that addition of peptide mixtures to culture media gives a further 5-fold increase in transport of dipeptides and oligopeptides; the effect is specific for peptide transport, amino acid uptake being unaffected. Presence of peptides but not amino acids overrides NH4+ repression of peptide transport. Step-up activation of transport activity, caused by addition of peptides to incubation media, and step-down inhibition that accompanies removal of peptides, occurs rapidly (within 30 min at 28 degrees C). Step-up is independent of de novo protein synthesis. This substrate-induced regulation is compatible with a rapid, reversible activation of plasma membrane-bound peptide permease(s), or a mechanism of endocytosis involving a cycle of insertion and retrieval of preformed permease components. These results are considered in relation to the expression of peptide permeases in vivo, and the development of synthetic anticandidal peptide carrier prodrugs designed to exploit these systems.     

The effect of azaserine on glutamine uptake by rat renal brush-border membranes.

Azaserine added directly to isolated rat renal brush-border membrane vesicles inhibits uptake of L-glutamine. Azaserine acts as a non-competitive inhibitor of the low-Km system for glutamine transport, but has no effect on the high-Km system. Preincubation of the vesicles with azaserine at 37 degrees C min is not required for transport inhibition to occur, although it is a requirement for gamma-glutamyl transpeptidase inhibition. Removal of azaserine from the vesicle preparation by repeated resuspensions in buffer results in a reversal of the transport inhibition but not in reversal of enzyme inhibition. Azaserine also inhibits vesicle uptake of L-proline and alpha-methyl D-glucoside, indicating a generalized effect on membrane transport systems. The data cast doubt on the postulate that gamma-glutamyl transpeptidase might act as the carrier mechanism for glutamine reabsorption by renal cortical cells.     

The quinoid structure is the molecular requirement for recognition of phthaleins by the organic anion carrier at the sinusoidal plasma membrane level in the liver

Sulfobromophthalein electrogenic uptake into rat liver plasma membrane vesicles was shown to admit only the quinoid, trivalent anion. The minimum requirement for this electrogenic process has been investigated in rat liver plasma membrane vesicles by using Thymol blue, a pH-indicator phthalein occurring either as a neutral, phenolic molecule or as a quinoid, monovalent anion. It has been found that Thymol blue is taken up electrogenically, in accordance with Michaelis-Menten kinetics. Parallel inhibition experiments have shown that both sulfobromophthalein and Thymol blue electrogenic uptakes are performed by the same carrier. It is, therefore, concluded that the phthalein structure recognized for transport is the quinoid molecule, with the dissociated acidic function on the benzene ring. Moreover, inhibitions by rifamycin-SV and bilirubin suggest that there exists a common uptake system for bilirubin, phthaleins and other anions. Taurocholate, on the contrary, does not appear to be involved in the same process.     

Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy. Function of bilitranslocase

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.