Báo cáo khoa học: Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy Function of bilitranslocase doc

Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy Function of bilitranslocase Sabina Passamonti 1 , Michela Terdoslavich 1 , Alja Margon 1,2 , Alessandra Cocolo 1 , Nevenka Medic 1 , Fulvio Micali 1 , Giuliana Decorti 3 and Mladen Franko 2 1 Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita ` di Trieste, Italy 2 Laboratory for Environmental Research, Nova Gorica Polytechnic, Slovenia 3 Dipartimento di Scienze Biomediche, Universita ` di Trieste, Italy Bilirubin-IXa is a lipophilic tetrapyrrole derived from heme catabolism in mammals [1]. In the plasma, it is transported as a reversible complex with serum albu- min, characterized by a dissociation constant in the range 10 )7 )10 )8 m [2,3]. The concentration of bilirubin in the plasma (0.3–1 mgÆ100 mL )1 ; 5–17 lm) results from a balance between its production from heme (mainly from hemoglobin) and its elimination into the bile. Hepatic disposal of bilirubin is an energy-depend- ent process, as it is first conjugated with glucuronic acid [4] by UDP-glucuronyl transferase 1 [5], then actively excreted into the biliary tract. The latter is a rate-limiting step [6], catalysed by the primary active transporter MRP2 [7], driving the overall flux of biliru- bin from the blood into the bile. The transport of bilirubin from the blood to the liver is a carrier-mediated mechanism, shared with both bromosulfophthalein (BSP) and indocyanine green [8]. This generated the working hypothesis that the bilirubin carrier could be identified, and possibly isolated, by applying its property to bind and transport BSP, instead of bilirubin. Initially, by applying an assay of BSP bind- ing, some proteins were isolated from liver plasma mem- brane fractions, such as bilitranslocase [9], the organic anion binding protein [10] and the BSP ⁄ bilirubin-bind- ing protein [11]. Later, by application of the functional Keywords bilirubin; bilitranslocase; HepG2 cells; thermal lens spectrometry; uptake assay Correspondence S. Passamonti, Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole, Universita ` di Trieste, via L. Giorgeri 1, I-34127 Trieste, Italy Fax: +39 040 558 3691 Tel: +39 040 558 3681 E-mail: passamonti@bbcm.units.it URL: http://www.bbcm.units.it (Received 28 July 2005, revised 18 August 2005, accepted 30 August 2005) doi:10.1111/j.1742-4658.2005.04949.x 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 activ- ity 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  50 nm in the absence of albumin. To ana- lyse the relative changes in bilirubin concentration in the medium through- out 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 indo- cyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bili- rubin and bromosulfophthalein, but not for indocyanine green. Abbreviations BSP, bromosulfophthalein; ICG, indocyanine green; Oatp, organic-anion-transporting polypeptide; PMSF, phenylmethanesulfonyl fluoride. 5522 FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS expression cloning technique, itself based on an assay of membrane transport of BSP [12], the first organic- anion-transporting polypeptide (Oatp) was isolated from rat liver [13]. Further congeners of these polypep- tides, all belonging to a single-gene superfamily, were identified in both rat and human tissues and found to mediate the membrane transport of diverse substrates, including BSP, bile salts, drugs and toxins [14]. A role as bilirubin carriers has been recently proposed for some of them, such as the human members OATP1B1 and OATP1B3, on the basis of various experimental obser- vations, both direct and indirect [15–20]. It has been calculated that about 0.25 g bilirubin per day is transported from the blood into the liver [1]. Fine regulation of this step is probably achieved through the expression of more than one type of bili- rubin carrier, as originally inferred from in vivo obser- vations in the rat [8]. A major property of rat liver bilitranslocase, a BSP carrier [21,22], is its ability to bind bilirubin, forming a complex with dissociation constant  2nm. Interest- ingly, this property has been reported in two previous studies on rat liver plasma membrane vesicles, where the protein occurs in its native environment and is assayed as an electrogenic BSP carrier [23,24]. The high affinity of bilitranslocase for bilirubin points to a possible inter- action with the albumin-free fraction of plasma bili- rubin, the concentration of which is < 10 )7 m [25]. The aim of this work was to investigate if the uptake of bilirubin in isolated liver cells requires the activity of bilitranslocase. An assay of bilirubin uptake by HepG2 cell cultures was set up, using an albumin-free transport medium containing 50 nm bilirubin. Thermal lens spectrometry was used to analyse these very dilute solutions of bilirubin, as the limit of detection of ana- lytes is 100-fold lower than with conventional spectro- photometry [26,27]. The human hepatocarcinoma cell line HepG2 provided the experimental cell model. The data obtained indicate that a carrier with the func- tional properties of bilitranslocase controls the per- meability of HepG2 cells to bilirubin. Results and Discussion Expression of bilitranslocase in HepG2 cells HepG2 cells were used to study hepatic uptake of bili- rubin as they are of human origin [28], they can be easily handled, and they retain various cellular func- tions typical of normal liver [29]. An early study, based on both immunological and functional analysis, repor- ted that bilitranslocase is expressed in HepG2 cells [30]. In addition, HepG2 cells express other putative bilirubin carriers, such as OATP1B1 and OATP1B3, to a very low level, if at all [31]. These properties simplify the interpretation of the experimental results. To confirm the presence of bilitranslocase in this cell model, a postmitochondrial fraction was isolated from a HepG2 homogenate, and the resulting proteins were separated by SDS ⁄ PAGE. Bilitranslocase was detected by immunoblot analysis as a band with electrophoretic mobility close to 38 kDa (Fig. 1A). The antibody used was an anti-sequence anti-bilitranslocase, prepared as described previously [24]. Electrogenic BSP uptake in HepG2 membrane vesicles The membrane fraction obtained from HepG2 cells was also tested for bilitranslocase-specific transport activity, assayed as electrogenic (valinomycin-induced) BSP transport, as described previously [32,33]. This Fig. 1. (A) Immunoblot of postmitochondrial fractions obtained from either rat liver or HepG2 cells. Samples were separated by SDS ⁄ PAGE and transferred to a nitrocellulose membrane. The blot was developed with a bilitranslocase antibody raised in rabbit (antibody A) as the pri- mary antibody. The secondary antibody was an anti-rabbit IgG conjugated with alkaline phosphatase. The membrane was stained by addition of bromochloroindolyl phosphate and nitroblue tetrazolium. Lane 1, erythrocyte ghosts (negative control); lane 2, rat liver; lane 3, HepG2 cells. Further details are described in Experimental procedures. (B) Immunogold particles visualized by scanning electron microscopy of a sector of a HepG2 cell. HepG2 cells were preincubated with an anti-sequence anti-bilitranslocase IgG (antibody A) raised in rabbit. The primary immunocomplexes were detected by the formation of secondary immunocomplexes, using colloidal gold (20 nm)-conjugated anti-rabbit IgGs. Gold particles are clearly visible as bright spots. S. Passamonti et al. Bilirubin uptake into HepG2 cells FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS 5523 activity was found to depend on substrate concentra- tion, in accordance with the Michaelis–Menten equa- tion. The derived K m value (3.55 ± 0.26 lm BSP) was similar to that of analogous fractions obtained from rat liver [33]. Moreover, these data were in close agree- ment with the value derived from experiments of BSP uptake in intact HepG2 cells (3.6 ± 1 lm) [30]. To check whether the electrogenic BSP uptake activity was related to bilirubin, the kinetics of BSP uptake in the presence of bilirubin was examined. The pigment acted as a competitive inhibitor (K i ¼ 116 ± 7 nm) with respect to BSP. These results agree with those previously obtained with rat liver plasma membrane vesicles [34], suggesting both a strong functional simi- larity of the rat and human homologues of bilitrans- locase and the involvement of the BSP electrogenic carrier in bilirubin binding and transport. Anti-sequence antibodies as tools to establish the role of bilitranslocase in organic anion uptake in HepG2 cells The uptake of polar solutes into cells is based on the activity of membrane carriers [35]. Once dissolved in aqueous solution (pH 7.4) up to about 50 nm, bilirubin may occur in solvated metastable aggregates [36], obvi- ously in equilibrium with solvated monomers. Under these conditions, monomeric bilirubin is the only spe- cies presumably taken up into liver cells by a carrier- mediated mechanism. When bilitranslocase was assayed as the carrier catalysing the electrogenic BSP uptake in rat liver plasma membrane vesicles, it was shown to be inhibited by an anti-sequence antibody, targeting segment 65–75 (EDSQGQHLSSF) of its pri- mary structure [24]. From the effect of bilirubin on the antibody inhibition kinetics, it was concluded that this antibody had targeted a high-affinity binding site of the electrogenic BSP carrier (K d of the carrier-bilirubin complex ¼ 2nm) [24]. Another anti-sequence antibody, targeting segment 235–246 (EFTYQLTSSPTC) of the primary structure of bilitranslocase, was recently shown to have similar effects on the electrogenic BSP uptake in rat liver plasma membrane vesicles [34]. This antibody was shown to interact with a distinct bilirubin-binding domain, characterized by even higher affinity for bilirubin (K d ¼ 0.33 nm) [34]. For the sake of clarity, the two antibodies are referred to as antibody A (anti-65–75) and antibody B (anti-235–246). The ability of these antibodies to form immuno- complexes on the extracellular surface of liver cells was checked by identifying the primary immunocomplexes with colloidal gold-conjugated secondary antibodies, visualized by scanning electron microscopy. Figure 1B shows a scanning electron micrograph of the surface of a HepG2 cell. Colloidal gold particles appear as white spots, locating the epitope of the bilitranslocase targeted by antibody A. Similar results were also obtained in primary rat hepatocytes and again in both types of cell using antibody B (not shown). Moreover, both antibodies were shown to inhibit the uptake into HepG2 cells of BSP [37], malvidin 3-gluco- side [37], and other newly identified competitive inhibi- tors of bilitranslocase (M. Terdoslavich, unpublished data). Therefore it appears that both antibodies not only bind to, but also partially impair, the bilitranslo- case function when assayed in intact cells, making them useful tools for studying the mechanism of bili- rubin uptake into HepG2 cells. Bilirubin analysis by thermal lens spectroscopy To obtain a calibration curve and determine the limit of detection of thermal lens spectrometry, serial solu- tions of bilirubin ranging from 2 to 50 nm were pre- pared in NaCl ⁄ P i . Before the measurement, the solutions were diluted 1 : 1 (v ⁄ v) with methanol to improve the thermo-optical properties of the samples (higher temperature coefficient of the refractive index, lower thermal conductivity) and thus to increase the sensitivity of the method. To avoid substantial photo- degradation from the excitation laser beam (476 nm, 120 mW), readings of thermal lens spectrometry sig- nals were taken within the first minute after insertion of the sample cell into the instrument. During this time interval, the decrease in the signal was less than 5%, which is a typical maximal relative error of the thermal lens spectrometry technique. Figure 2 shows that there is good correlation between the concentration of bili- rubin and the detected signal. From the regression line, it can be assumed that the limit of detection is some- where between 1 and 2 nm. Thermal lens spectrometry hence appears to be suitable for the detection of bili- rubin in albumin-free physiological solutions. To check for any possible contribution to the chan- ges in absorbance from the plastic-ware, appropriate blank samples were analysed. No changes in the back- ground signal were observed with time. Assay of bilirubin uptake into HepG2 cells Cells were grown to confluence in 25 cm 2 flasks. Before the assay, the cell growth medium was removed and the monolayer rinsed three times with 5 mL NaCl ⁄ P i . Then, the transport medium, consisting of Bilirubin uptake into HepG2 cells S. Passamonti et al. 5524 FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS 7 mL NaCl ⁄ P i containing 50 nm bilirubin, was added to the cell monolayer. Samples of the transport med- ium were withdrawn at time intervals and delivered to conical tubes containing an equal volume of methanol. The tubes were centrifuged and the supernatants ana- lysed by thermal lens spectroscopy within the same day. It was expected that the uptake of bilirubin into the cells would result in a decrease in the initial bili- rubin concentration in the assay medium. Figure 3 shows the results of an experiment to examine the uptake of bilirubin at three concentrations (10, 30 and 50 nm) by HepG2 cells. In the absence of the pigment, the signal was stable, showing that the cell monolayer did not release compounds, such as carotenoids or fla- vins, that might interfere with the spectroscopic analy- sis. The data show that the signal was stable even in the presence of bilirubin. This finding, although unexpected, ruled out the possibility that bilirubin may bind unspecifically to the cell surface or that it could be destroyed during the experiment. On the other hand, it also suggested that these cells either did not take up the pigment or, less likely, did not retain it inside the cytoplasm. A third possibility is that carriers for bilirubin were either absent or in an inactive state under the experimental conditions. On the assumption that one of the bili- rubin carriers may be bilitranslocase, we attempted to increase its transport activity. In isolated rat liver plasma membrane vesicles, this carrier has been shown to occur in a metastable equilib- rium of two functional states, characterized by either high (C conformer) or low (C* conformer) affinity for the substrate BSP [33]. This equilibrium is regulated, in vitro, by the concentration of certain substrates, such as BSP itself and nicotinic acid. It was speculated, how- ever, that other allosteric effectors, possibly resulting from intracellular metabolism, may regulate the overall activity of bilitranslocase [33]. Data from this laborat- ory (S. Passamonti, unpublished data) clearly show that the redox equilibrium of the nicotinamide nucleotides modulates the allosteric equilibrium of bilitranslocase. In particular, a low NADH ⁄ NAD + ratio, such as that occurring in physiological conditions [38], favours the low-affinity state. An analogous allosteric equilibrium of the bilitran- slocase homologue may occur in HepG2 cells. Thus, increasing the relative concentration of NADH in the cytoplasm may activate bilitranslocase. This could be achieved by preincubating the cell monolayer in the presence of 5 mm lactate for 1 h. In the cells, lactate oxidizes to pyruvate, lowering the NAD + ⁄ NADH ratio. Under these conditions, the bilirubin concentra- tion in the cell medium was found to decrease by  40% within 100 s at 37 °C, but not at 0 °C (Fig. 4, inset), which is probably an effect of a temperature- dependent uptake into the cell monolayer. A similar uptake could also be observed by replacing lactate with 5 mm ethanol, another NADH-generating Fig. 2. Calibration curve of bilirubin analysed by thermal lens spectro- metry. A series of bilirubin solutions was prepared by appro- priately diluting a bilirubin stock (10 l M in dimethylsulfoxide) in NaCl ⁄ P i ⁄ methanol (1 : 1, v ⁄ v). Samples (1.2 mL) were added to the spectrophotometric cuvette and analysed by thermal lens spectro- metry, with excitation laser operating at 120 mW power and 476 nm wavelength. Data (n ¼ 3) are means ± SEM and were fit- ted to the y ¼ y 0 + mx equation. The following parameters were obtained: y 0 ¼ 0.6414, m ¼ 0.0939, r 2 ¼ 0.9915. Fig. 3. Thermal lens spectrometry signal of bilirubin solutions applied to HepG2 monolayers. Monolayers of HepG2 cells grown in 25-cm 2 flasks were exposed to 7 mL of the NaCl ⁄ P i solution in either the absence (d) or presence of 10 n M (s), 30 nM (m)or 50 n M (n) bilirubin. Samples were withdrawn at the indicated times, processed and analysed as described in Experimental procedures. All procedures were carried out at 37 °C. Data are mean ± SEM (n ¼ 4) and were fitted to the y ¼ y 0 + mx equation. S. Passamonti et al. Bilirubin uptake into HepG2 cells FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS 5525 substrate. Figure 4 shows the data from three separate experiments, each carried out in quadruplicate. Thus, the increased permeability of HepG2 cells to bilirubin may be due to either a direct, activating effect on bilirubin membrane carriers or an increased ability of the cells to accumulate bilirubin. In either case, we concluded that the conditions for assaying bilirubin uptake by the cells must include a 1-h preincubation in the presence of lactate (or ethanol) to decrease the intracellular NAD + ⁄ NADH ratio. Bilirubin uptake into HepG2 cells: effect of anti-sequence bilitranslocase antibodies To examine whether the uptake of bilirubin can be accounted for by the activity of bilitranslocase, cells were preincubated with antibody A, added to fresh, serum-free growth medium containing 5 mm lactate for 1 h before the transport experiment. Figure 5 shows that cells lost the ability to take up bilirubin. It is worth noting that essentially no free immunoglobulins were present in the transport medium, as cells had been extensively rinsed before the addition of bilirubin. The effect observed is therefore due to the formation of stable immunocomplexes on the monolayer’s sur- face. In a separate experiment, it was confirmed that unspecific immunoglobulins had no influence on bili- rubin uptake (Fig. 5, inset). When antibody B was used, no effect on the bili- rubin uptake was observed (Fig. 5, inset). This result, although unexpected, is consistent with the view that the protein segment targeted by this antibody may not be involved in the translocation of bilirubin by the car- rier. This result also shows the high specificity of the biological action of antibody A, together with the absence of effects by unspecific IgGs. It can be conclu- ded that bilirubin uptake into HepG2 cells depends on the activity of a membrane carrier. Bilirubin uptake into HepG2 cells: effect of a protein-modifying reagent We attempted to disrupt the integrity of the bilirubin carrier by means of the protein-modifying reagent phenylmethanesulfonyl fluoride (PMSF). This serine- specific reagent was used because it had been shown to inhibit electrogenic BSP uptake in rat liver plasma membrane vesicles [23]. A crucial observation was that bilirubin and nicotinic acid could both prevent this inhibition. In both cases, the protection dis- played a hyperbolic concentration dependence, with Fig. 4. Bilirubin uptake into HepG2 monolayers: effect of reducing substrates in the preincubation and of the assay temperature. Monolayers of HepG2 cells grown in 25-cm 2 flasks were preincu- bated for 1 h in the presence of either 5 m M lactate (squares and triangles) or 5 m M ethanol (circles). After removal of the culture medium, cells were washed and exposed to 7 mL NaCl ⁄ P i solution containing 50 n M bilirubin. Samples were withdrawn at the indica- ted times, processed and analysed as described in Experimental procedures. The uptake assay was carried out at 37 °C (circles and squares) and at 0 °C (triangles). Data are mean ± SEM (n ¼ 4). The inset shows the raw data obtained by thermal lens spectrometry. Error bars are not visible if smaller than symbols. Fig. 5. Bilirubin uptake into HepG2 monolayers: effect of antibody A in the preincubation. Monolayers of HepG2 cells grown in 25-cm 2 flasks were preincubated for 1 h in the presence of 5 mM lactate, either without (squares) or with (m) 0.25 lg antibody A per mL. After removal of the culture medium, cells were washed and exposed to 7 mL NaCl ⁄ P i solution containing 50 nM bilirubin. Sam- ples were withdrawn at the indicated times, processed and ana- lysed as described in Experimental procedures. The uptake assay was carried out at 37 ° C. Data are mean ± SEM (n ¼ 4). The inset shows the results obtained by preincubating HepG2 cell mono- layers in the absence (squares) or presence of rabbit immunoglobu- lins purified from preimmune sera (r) or antibody B (circles), both used as 0.25 lgIgGÆmL )1 . Error bars are not visible if smaller than symbols. Bilirubin uptake into HepG2 cells S. Passamonti et al. 5526 FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS half-maximal effects at 2 and 11 nm, respectively. Moreover, at saturating concentrations of the two ligands, electrogenic BSP uptake was fully refractory to PMSF [23]. Thus, it was inferred that PMSF targeted a specific site on the BSP electrogenic car- rier involved in the binding of both bilirubin and nicotinic acid. At that time, it was also speculated that bilirubin uptake into the liver may be impaired, if not blocked, by the chemical modification of serines of the BSP electrogenic carrier. This predic- tion was tested experimentally in the following experiment. HepG2 cells were preincubated with lactate as speci- fied above; 20 min before the end of the preincubation, 0.1 mm PMSF was added to the growth medium. Bili- rubin uptake was then assayed as described above. Figure 6 shows that the uptake of bilirubin was strongly inhibited under these conditions. Bilirubin uptake into HepG2 cells: effect of nicotinic acid, taurocholate and digoxin Previous work has shown that the inhibition of electro- genic BSP uptake into rat liver plasma membrane vesi- cles by either antibody A [24] or PMSF [23] is strongly influenced by both bilirubin and nicotinic acid, sug- gesting that bilirubin and nicotinic acid share a com- mon binding site on the carrier involved. To test this, bilirubin uptake in HepG2 cells was tested in the pres- ence of 1 lm nicotinic acid, a saturating concentration for bilitranslocase [23,24]. As shown in Fig. 7, bilirubin uptake was completely blocked under these conditions, probably as a result of competition at the level of the bilirubin carrier, rather than due to intracellular bilirubin binding and conju- gation, as these two steps have not been documented in the hepatic metabolism of nicotinic acid [39]. The effects of taurocholate and digoxin were also examined. These compounds have been shown not to inhibit bilitranslocase transport activity in rat liver plasma membrane vesicles (data not shown and [40]), but are well-known substrates of OATP carriers. In particular, it was expected that taurocholate would inhibit both OATP carriers expressed in HepG2, i.e. OATP1A2 (OATP-A) and OATP1B3 (OATP8) [31], whereas digoxin would inhibit only OATP1B3 [41]. This test is important because the latter has been shown to transport bilirubin [18]. As shown in Fig. 7, 100 lm taurocholate did not influence bilirubin uptake, whereas 2 lm digoxin was found to delay the onset of bilirubin uptake. We are unclear about the biological meaning of these results, because, had OATP1B3 been a digoxin-sensitive bili- rubin carrier, it would have been inhibited by tauro- cholate as well. Fig. 6. Bilirubin uptake into HepG2 monolayers: effect of PMSF in the preincubation. Monolayers of HepG2 cells grown in 25-cm 2 flasks were preincubated for 1 h in the presence of 5 mM lactate, without (squares) or with (m) 0.1 m M PMSF added 20 min before the end of the preincubation. After removal of the culture medium, cells were washed and exposed to 7 mL NaCl ⁄ P i solution contain- ing 50 n M bilirubin. Samples were withdrawn at the indicated times, processed and analysed as described in Experimental proce- dures. The uptake assay was carried out at 37 °C. Data are mean ± SEM (n ¼ 4). Error bars are not visible if smaller than symbols. Fig. 7. Bilirubin uptake into HepG2 monolayers: effects of the addi- tion of nicotinic acid, taurocholate and digoxin. Monolayers of HepG2 cells grown in 25-cm 2 flasks were preincubated for 1 h in the presence of 5 m M lactate. After removal of the culture med- ium, cells were washed and exposed to 7 mL NaCl ⁄ P i solutions containing 50 n M bilirubin in the absence (squares) or presence of either 1 l M nicotinic acid (m) or 100 lM taurocholate (d)or2lM digoxin (.). Samples were withdrawn at the indicated times, proc- essed and analysed as described in Experimental procedures. The uptake assay was carried out at 37 °C. Data are mean ± SEM (n ¼ 4). Error bars are not visible if smaller than symbols. S. Passamonti et al. Bilirubin uptake into HepG2 cells FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS 5527 BSP uptake into HepG2 cells: effect of bilirubin, nicotinic acid and taurocholate The uptake of bilirubin and BSP from the blood into the liver has long been known to occur by an appar- ently common mechanism of transport [8,42]. To test this concept in HepG2 cells, BSP uptake was studied using an identical experimental approach to the bili- rubin-uptake experiments, i.e. by measuring the time course of disappearance of the substrate from the extracellular medium. The latter was interpreted as uptake of BSP into the cell monolayer and plotted accordingly against time, as in a previous study [37]. Figure 8 shows that BSP uptake increased rapidly with time and reached the steady state in  30 s. In the presence of 100 lm taurocholate, both the rate and extent of BSP uptake were unchanged, suggesting that bile salt carriers are not involved in the event. In con- trast, the figure shows that both 50 nm bilirubin and 1 lm nicotinic acid inhibited BSP uptake; interestingly, both substrates inhibited only the onset of BSP uptake, which was slightly delayed, in analogy with the effects produced by the antibodies to bilitranslocase [37]. Thus, it can be concluded that BSP uptake into HepG2 cells is partially accounted for by the activity of a carrier of bilirubin and nicotinic acid, presumably bilitranslocase. Indocyanine green (ICG) uptake into HepG2 cells: effects of PMSF and antibodies to bilitranslocase Another anionic dye, ICG, is also known to be taken up by the liver via a carrier-mediated mechanism, shared with both BSP and bilirubin [8]. When tested as a reversible inhibitor of electrogenic BSP uptake in rat liver plasma membrane vesicles, it displayed an unusual effect, consisting of a sharp increase in the initial rate of uptake, such that it could not be reliably measured (data not shown). This precluded the kinetic characteri- zation of its effect. Therefore, to investigate the poss- ible involvement of bilitranslocase in ICG transport, the uptake of this dye into HepG2 cells was examined. Figure 9 shows the rapid uptake of 1.5 lm ICG in HepG2 cells and its temperature-dependence. Neither antibody A nor PMSF had any effect on the time course of the dye uptake. Similar results were also obtained at a higher ICG concentration (6 lm, not shown). ICG uptake into HepG2 cells: effects of substrates specific for either bilitranslocase or OATP carriers Figure 10 shows the results obtained by adding various organic anions to the ICG solution. Figure 10A shows that the time course of the dye uptake in the presence Fig. 8. BSP uptake by HepG2 monolayers: effects of the addition of bilirubin, nicotinic acid and taurocholate. Monolayers of HepG2 cells were grown in 25-cm 2 flasks. After removal of the culture medium, cells were washed and exposed to 3.5 mL NaCl ⁄ P i solu- tion containing 24 l M BSP in the absence (squares) or presence of 50 n M bilirubin (s)or1lM nicotinic acid (n) or 100 l M taurocholate (.). Samples were withdrawn at the indicated times, processed and analysed as described in Experimental procedures. The uptake assay was carried out at 37 °C. Data are mean ± SEM (n ¼ 4). Error bars are not visible if smaller than symbols. Fig. 9. ICG uptake by HepG2 monolayers: effect of either antibody A or PMSF in the preincubation. Monolayers of HepG2 cells grown in 25-cm 2 flasks were p reincubated for 20 min in the absence (cir- cles and diamonds) or presence of 0.25 lg antibody AÆml )1 (.)or 0.1 m M PMSF (m). After removal of the cell culture medium, cells were washed and exposed to 7 mL NaCl ⁄ P i solution containing 1.5 l M ICG. Samples were withdrawn at the indicated times, proc- essed and analysed as described in Experimental procedures. The uptake assay was carried out at 37 °C, in all cases, except for a control (diamond) carried out at 0 °C. Data are mean ± SEM (n ¼ 4). Error bars are not visible if smaller than symbols. Bilirubin uptake into HepG2 cells S. Passamonti et al. 5528 FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS of 1 lm nicotinic acid was identical with that of the control. In the same set of tests, 50 nm bilirubin had only a small inhibitory effect on the late phase of the uptake. In contrast, both taurocholate (100 lm) and digoxin (2 lm) inhibited the overall time course of the dye uptake (Fig. 10B). As digoxin is a specific substrate of OATP1B3, this result suggests that this carrier, although expressed to a limited extent in this cell line [31], may also be involved in ICG uptake. However, as the inhibition caused by this compound was only par- tial, we cannot conclude whether it is due to nonsatu- rating concentrations of this inhibitor or to the activity of other unknown digoxin-insensitive ICG carriers. Conclusions and perspectives Bilirubin uptake in HepG2 cells is a carrier- mediated event The possibility of using thermal lens spectroscopy for determining bilirubin concentration in aqueous solu- tions, within the limits of its solubility at physiological pH, has opened up the unprecedented opportunity to examine its cellular uptake from a solvent-free and albumin-free solution. Under these conditions, no unspecific adsorption of the pigment to the cell surface could be detected. Indeed, we observed that the pig- ment concentration in the assay medium remained constant: (a) in all cases (Fig. 3) except when the cells were preincubated in the presence of reducing sub- strates (Fig. 4); (b) when the assay was carried out on ice, even with cells preincubated in the presence of reducing substrates (Fig. 4); (c) when the assay med- ium contained nicotinic acid, even with cells preincu- bated in the presence of reducing substrates (Fig. 7); and (d) when the cells were preincubated in the presence of reducing substrates and the protein-modi- fying reagent PMSF (Fig. 6). Thus, the apparent disappearance of bilirubin from the extracellular medium was assumed to be due to cel- lular uptake. The latter is clearly a carrier-mediated event, as it was not only temperature sensitive (Fig. 4), but also greatly and specifically reduced by a reversible inhibitor, such as nicotinic acid (Fig. 7), by a covalent protein-modifying reagent (Fig. 6) and by a bilitrans- locase antibody (Fig. 5). Under the prevailing assay conditions,  90 pmol bilirubin disappeared from the medium and were accu- mulated in the cell monolayer. Applying the typical ratio of 7 lL per mg protein to the 3 mg protein of the HepG2 monolayer, the monolayer volume can be estimated to be  20 lL. The intracellular bilirubin concentration can thus be estimated to be 4.5 lm, i.e. 90 times higher than the extracellular concentration. The rate of bilirubin uptake observed in this assay is  5 pmolÆmin )1 Æ10 )6 cells. Assuming that the initial rate of uptake is directly proportional to the number of cells, it can be estimated that 0.75 lmol bilirubinÆmin )1 could be extracted by 1.5 · 10 11 cells, the estimated number of cells in a normal human liver. This corres- ponds to the extraction of about 1 mmol per day, i.e. 0.58 g per day. This value is slightly higher than the physiological flux of bilirubin from the blood into the liver (0.25 g per day [1]), which is itself rate-limited by the canalicular excretion step. The main bilirubin carrier in HepG2 cells has the functional features of the electrogenic BSP carrier in rat liver plasma membrane vesicles Various pieces of evidence consistently indicate that the functional features of bilirubin uptake in HepG2 Fig. 10. ICG uptake by HepG2 monolayers: effects of either nicotinic acid and bilirubin (A) or taurocholate and digoxin (B). Monolayers of HepG2 cells were grown in 25-cm 2 flasks. After removal of the culture medium, cells were washed and exposed to 7 mL NaCl ⁄ P i solution containing 1.5 l M ICG in the absence (squares, in both panels) or presence of 50 nM bilirubin (s,A), 1 lM nicotinic acid (e, A), 100 lM tauro- cholate (.,B)or2l M digoxin (m, B). Samples were withdrawn at the indicated times, processed and analysed as described in Experimental procedures. The uptake assay was carried out at 37 °C. Data are mean ± SEM (n ¼ 4). Error bars are not visible if smaller than symbols. S. Passamonti et al. Bilirubin uptake into HepG2 cells FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS 5529 cells, shown in this study, closely match some of the functional features of electrogenic BSP uptake in rat liver plasma membrane vesicles. First, the electrogenic BSP uptake in rat liver plasma membrane vesicles can be inhibited by the serine-speci- fic reagent PMSF [23]. Complete protection against such inhibition can be yielded by both bilirubin and nicotinic acid at nanomolar concentrations. Kinetic analysis of these effects has enabled the calculation of the dissociation constants of the complexes of bilitrans- locase with the two ligands (2 and 11 nm, respectively), suggesting that the electrogenic BSP carrier is a high- affinity binding protein for those molecules [23]. The above results show that the bilirubin carrier in HepG2 cells is also sensitive to both PMSF and nicotinic acid. Moreover, the almost complete blockade of bilirubin uptake in HepG2 cells by PMSF contrasts with the only partial (no more than 30%) inhibition of electro- genic BSP uptake by the latter. This is in line with the earliest prediction, that the occupation of the bilitrans- locase bilirubin-binding site by either PMSF (by cova- lent binding to serines) or nicotinic acid (by reversible interaction) would totally impair bilirubin uptake in intact cells [23]. Second, the electrogenic BSP uptake in rat liver plasma membrane vesicles is inhibited by a polyclonal antibody raised against an undecapeptide correspond- ing to segment 65–75 of the primary structure of bili- translocase [24]. As in the case of PMSF, both bilirubin and nicotinic acid protected against such inhi- bition, yielding quite similar K d values for the com- plexes of bilitranslocase with these ligands. Third, the electrogenic BSP uptake is regulated by the NAD + ⁄ NADH ratio in a physiologically meaning- ful range (S. Passamonti, unpublished data). Crucial to the successful observation of bilirubin uptake in HepG2 cells was the choice of preincubating cells with reducing substrates such as lactate or ethanol. Collectively, these data suggest that the bilirubin carrier in HepG2 cells is quite similar to the electro- genic BSP carrier in rat liver plasma membrane vesi- cles. Therefore, this assay is indeed a reliable tool for investigating the transport of bilirubin and related substrates in the liver. The enigma of the primary structure of rat liver bilitranslocase and the biological activities of anti-sequence bilitranslocase antibodies The amino-acid sequence of bilitranslocase was origin- ally deduced from a clone selected from an expression library, on the basis of its ability to express a protein that reacted with a monoclonal antibody that inhibited electrogenic BSP uptake in rat liver plasma membrane vesicles [24]. Unfortunately, the clone was truncated, preventing its further characterization. The very high homology of the nucleotide sequence of the clone with that of the antisense strand of ceruloplasmin suggested that it was the product of a wrong cloning strategy. Nonetheless, it was noted that the translated primary structure of the clone contained a short segment (62–99) that was highly homologous to segment 6–45 of a number of a-chains of phycocyanins [24]. In this class of proteins, this segment is invariant and has been shown to be involved in accommodating the prosthetic group phyco- cyanobilin, an open tetrapyrrole [43]. Segment 62–99 of the clone was therefore expected to provide a poten- tial structural component for high-affinity bilirubin binding. This observation per se suggested investiga- tion of the occurrence of a protein encoded by the cloned sequence. Thus, the issue was examined experimentally using antibody A, which targets segment 65–75, and, under stringent conditions, reacts with: (a) a 38-kDa mem- brane protein in rat liver; (b) a 38-kDa protein purified using a standard protocol for the isolation of bilitrans- locase [44]; (c) the protein expressed by the clone in Escherichia coli [24]. Moreover, this antibody displayed a remarkable biological activity, in that it inhibited electrogenic BSP uptake in rat liver plasma membrane vesicles, by targeting a high-affinity binding site for both bilirubin and nicotinic acid [24]. In this work, the biological activity of this antibody is fully confirmed. Not only does it identify a homo- logue of rat liver bilitranslocase in the plasma mem- brane of HepG2 cells, but also it shows that the homologue in question is indeed a bilirubin carrier. To test further the existence of a liver membrane protein with the trait of the primary structure of bili- translocase, a second polyclonal anti-sequence anti- body (antibody B) was produced and found to have similar biological properties to those of the first [34].This antibody was, however, found to be ineffec- tive in inhibiting bilirubin uptake in HepG2 cells. This shows that although the segment targeted by antibody B (EFTYQLTSSPTC) binds bilirubin with extraordinarily high affinity [34], it does not disturb the transport of bilirubin from its surface binding site, which is targeted by antibody A, into the hepatocyte. This lack of inhibition is an exception, as the hepatocellular uptake of both BSP and malvidin 3-glucoside [37], as well as that of two recently identi- fied competitive inhibitors of electrogenic BSP uptake (M. Terdoslavich, unpublished data), were indeed inhibited by antibody B. This would suggest that the Bilirubin uptake into HepG2 cells S. Passamonti et al. 5530 FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS multiple interactions of bilirubin with the carrier, which conceivably underlie the substrate translocation through the transport pore of the carrier, are some- what different from those of the other substrates. A similar conjecture has previously been reported, based on certain properties of electrogenic BSP uptake in rat liver plasma membrane vesicles [23]. Other carriers participate in organic anion uptake in HepG2 cells Antibody A is the most specific bilitranslocase inhib- itor and is shown above to inhibit quite strongly bilirubin uptake in HepG2 cells, suggesting that bilitranslocase is the main carrier involved. Both PMSF and nicotinic acid fully inhibited bilirubin uptake, suggesting that, although in principle not spe- cific just for bilitranslocase, they can impair a cellular function exclusively performed by bilitranslocase. The uptake of BSP into HepG2 cells was in fact only partially inhibited by nicotinic acid, leaving intact the activity of other putative BSP carriers. In an effort to identify the latter carriers, taurocholate was used, but no effect was observed. These results suggest that bile salt carriers take no part in BSP uptake in HepG2 cells, which is consistent with the finding that sodium- dependent bile salt uptake is absent from these cells and sodium-independent bile salt uptake is lower than in primary hepatocytes [45]. The residual BSP uptake activity, however, calls for the existence of so far unidentified BSP carriers, which are undoubtedly not involved in bile salt transport. The activity of the BSP ⁄ bilirubin binding protein has been documented in HepG2 cells [46] and may be very strongly involved in our assay of BSP uptake. The fact that bilirubin uptake is not inhibited by taurocholate suggests again that bile salt carriers are inactive. This conclusion is supported by the observa- tion that the uptake of ICG, a high-affinity substrate of OATP1B1 [15], is not affected by taurocholate. However, the partial inhibition of both bilirubin and ICG uptake by digoxin suggests that OATP1B3, although poorly expressed [31], may act as a carrier for both bilirubin and ICG in HepG2 cells. In conclusion, this work shows that bilitranslocase is involved in the uptake of both bilirubin and BSP, but not of ICG, in HepG2 cells. The activity of bilitrans- locase in this cell line, which is very similar to that observed in normal liver, has apparently not been lost as a result of transformation, unlike that of bile salt carriers. As a consequence, the occurrence of addi- tional bilirubin carriers in normal liver cannot be ruled out. Experimental procedures Cell culture Human hepatoblastoma HepG2 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in Eagle’s minimum essential med- ium supplemented with 10% (v ⁄ v) fetal bovine serum, 1mm sodium pyruvate, 100 UÆmL )1 penicillin and 100 lgÆmL )1 streptomycin. Cells were grown in a humid- ified incubator at 5% CO 2 ⁄ 95% air (v ⁄ v) at 37 °C and after 6 days harvested by exposure to 0.05% trypsin and 0.02% EDTA (all these reagents were purchased from Euroclone Ltd, Wetherby, Yorks., UK) for 5 min. Cells (8.4 · 10 5 cells, at a cell density of 1.2 · 10 5 cellsÆmL )1 ) were seeded in 25 cm 2 flasks. After 6 days, as cells reached confluence, the medium was replaced, and uptake experi- ments were performed on the following day. Postmitochondrial fraction of HepG2 homogenates This fraction collects the plasma membrane and micro- somes of the cell homogenate. Cells (3.6 · 10 8 ) were harves- ted by scraping the flask bottom, collected in 8 mL of an ice-cold solution (10 mm Hepes, 0.25 m sucrose, pH 7.4) and homogenized in a Dounce tube using a small clearance pestle. The homogenate was centrifuged at 700 g for 10 min. The supernatant was collected and centrifuged again at 100 000 g for 1 h. The pellet was resuspended in the above solution at a final protein concentration of  10 mgÆmL )1 and stored in aliquots at )80 °C. Bilitrans- locase transport activity was almost entirely recovered in this fraction. Assay of bilitranslocase transport activity Bilitranslocase transport activity was assayed spectrophoto- metrically as previously described in detail [32–34]. Briefly, 3–10 lL( 10 lg protein) of the postmitochondrial fraction was added to a stirred cuvette containing 2 mL assay med- ium (0.1 m potassium phosphate, pH 8.0, with 18 lm BSP, without or with 100 nm bilirubin as a reversible inhibitor) at room temperature. This addition caused an instantaneous fall in absorbance (recorded at k ¼ 580–514 nm). After the attainment of a steady-state (4 s), a second fall in absorb- ance was brought about by adding 5 lg valinomycin in 1 lL methanol, which was due to valinomycin-induced K + diffusion potential. This K + diffusion drove the substrate into the vesicles [32]. The slope of the linear phase of this decrease in absorbance, lasting about 1 s, is referred to as electrogenic BSP uptake and is related to bilitranslocase transport activity [24,47]. The pH in the assay medium was constant throughout the test, as previously shown with an analogous preparation from rat liver [32]. S. Passamonti et al. Bilirubin uptake into HepG2 cells FEBS Journal 272 (2005) 5522–5535 ª 2005 FEBS 5531 [...]... by a Fourier transformation of the signal while filtering out all other frequencies Assay of BSP and ICG uptake in HepG2 cells On the day of the experiment, the cells were preincubated for 20 min with 2.5 mL fresh medium, in either the absence or presence of bilitranslocase antibodies (0.25 lg IgGÆmL)1) Then, the medium was removed, and the cells were washed four times with NaCl ⁄ Pi, at 37 °C The uptake. .. corresponding to segment 235–246 of the primary structure of bilitranslocase The peptide was conjugated to maleimideactivated keyhole limpet hemocyanin (Antibody production and purification kit; Pierce, Rockford, IL, USA) and injected into a rabbit; sera were purified by affinity chromatography as previously described [34] Assay of bilirubin uptake in HepG2 cells On the day of the experiment, the cell medium... until analysis by thermal lens spectroscopy on the same day, as described below All experiments were performed in quadruplicate S Passamonti et al The flasks were kept in a water bath at 37 °C and shaken as above Samples were collected at time intervals and transferred to polycarbonate spectrophotometric cuvettes for analysis Thermal lens spectrometric analysis of bilirubin The thermal lens spectrometric.. .Bilirubin uptake into HepG2 cells Antibodies to bilitranslocase Antibody A was raised in one rabbit (Oryctolagus cuniculus, New Zealand White strain), immunized with a multiantigen peptide-based system as described [24], using the peptide EDSQGQHLSSF, corresponding to segment 65–75 of the primary structure of bilitranslocase Sera were purified by affinity chromatography as... 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Fund, Regione Friuli Venezia Giulia and Italian Ministry of Welfare) are acknowledged References 1 McDonagh AF (2001) Turning green to gold Nat Struct Biol 8, 198–200 2 Petersen CE, Ha CE, Harohalli K, Feix JB & Bhagavan NV (2000) A dynamic model for bilirubin binding to human serum albumin J Biol Chem 275, 20985–20995 5533 Bilirubin uptake into HepG2 cells 3 Weisiger RA, Ostrow JD, Koehler RK, Webster... mode-mismatched thermal lens spectrometer [27] schematically shown in Fig 11 The excitation source (pump laser beam) was provided by an argon ion laser (Coherent; Innova 90, Santa Paula, CA, USA), tuned to a 476-nm line (120 mW), and modulated by a mechanical chopper (Scitec Instruments, Redruth, Cornwall, UK) at 10 Hz The pump beam was focused on to the sample cell by a 250-mm focal length lens A second lens . Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy Function of bilitranslocase Sabina Passamonti 1 ,. per- meability of HepG2 cells to bilirubin. Results and Discussion Expression of bilitranslocase in HepG2 cells HepG2 cells were used to study hepatic uptake of bili- rubin