uptake of bilirubin into hepg2 cells assayed by thermal lens spectroscopy : function of...

Uptake of bilirubin into HepG2 cells assayed by thermallens spectroscopy

Function of bilitranslocase

Sabina Passamonti1, Michela Terdoslavich1, Alja Margon1,2, Alessandra Cocolo1, Nevenka Medic1,Fulvio Micali1, Giuliana Decorti3 and Mladen Franko2

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: [email protected]

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 � 2 nm. 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 ⁄ PAGEand 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 Km 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 (Ki 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 (Kd of the carrier-bilirubin

complex ¼ 2 nm) [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 (Kd 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 ⁄Pi. 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 cm2 flasks.

Before the assay, the cell growth medium was removed

and the monolayer rinsed three times with 5 mL

NaCl ⁄Pi. Then, the transport medium, consisting of

Bilirubin uptake into HepG2 cells S. Passamonti et al.


7 mL NaCl ⁄Pi 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 lM in dimethylsulfoxide) in

NaCl ⁄ Pi ⁄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 y0 + mx equation. The following parameters were

obtained: y0 ¼ 0.6414, m ¼ 0.0939, r2 ¼ 0.9915.

Fig. 3. Thermal lens spectrometry signal of bilirubin solutions

applied to HepG2 monolayers. Monolayers of HepG2 cells grown in

25-cm2 flasks were exposed to 7 mL of the NaCl ⁄ Pi solution in

either the absence (d) or presence of 10 nM (s), 30 nM (m) or

50 nM (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 y0 + mx equation.



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-cm2 flasks were preincu-

bated for 1 h in the presence of either 5 mM lactate (squares and

triangles) or 5 mM ethanol (circles). After removal of the culture

medium, cells were washed and exposed to 7 mL NaCl ⁄ Pi solution

containing 50 nM 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-cm2 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 ⁄ Pi 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 lg IgGÆmL)1. Error bars are not visible if smaller than

symbols.



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-cm2

flasks were preincubated for 1 h in the presence of 5 mM lactate,

without (squares) or with (m) 0.1 mM 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 ⁄ Pi solution contain-

ing 50 nM 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-cm2 flasks were preincubated for 1 h in

the presence of 5 mM lactate. After removal of the culture med-

ium, cells were washed and exposed to 7 mL NaCl ⁄ Pi solutions

containing 50 nM bilirubin in the absence (squares) or presence of

either 1 lM nicotinic acid (m) or 100 lM taurocholate (d) or 2 lM

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 ¼4). Error bars are not visible if smaller than symbols.



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-cm2 flasks. After removal of the culture

medium, cells were washed and exposed to 3.5 mL NaCl ⁄ Pi solu-

tion containing 24 lM BSP in the absence (squares) or presence of

50 nM bilirubin (s) or 1 lM nicotinic acid (n) or 100 lM 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-cm2 flasks were preincubated for 20 min in the absence (cir-

cles and diamonds) or presence of 0.25 lg antibody AÆml)1 (.) or

0.1 mM PMSF (m). After removal of the cell culture medium, cells

were washed and exposed to 7 mL NaCl ⁄ Pi solution containing

1.5 lM 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 ¼4). Error bars are not visible if smaller than symbols.



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)1Æ10)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 · 1011 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-cm2 flasks. After removal of the culture medium, cells were washed and exposed to 7 mL NaCl ⁄ Pi solution

containing 1.5 lM 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) or 2 lM 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.



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 Kd 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



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,

1 mm sodium pyruvate, 100 UÆmL)1 penicillin and

100 lgÆmL)1 streptomycin. Cells were grown in a humid-

ified incubator at 5% CO2 ⁄ 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 · 105 cells, at a cell density of 1.2 · 105 cellsÆmL)1)

were seeded in 25 cm2 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 · 108) 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].



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 described previously [24]. Anti-

body B was obtained using peptide EFTYQLTSSPTC, cor-

responding to segment 235–246 of the primary structure of

bilitranslocase. The peptide was conjugated to maleimide-

activated keyhole limpet hemocyanin (Antibody production

and purification kit; Pierce, Rockford, IL, USA) and injec-

ted into a rabbit; sera were purified by affinity chromato-

graphy as previously described [34].

Assay of bilirubin uptake in HepG2 cells

On the day of the experiment, the cell medium was

replaced, and a 1 h preincubation at 37 �C was started by

adding 7 mL serum-free fresh medium containing 5 mm lac-

tic acid, or 5 mm ethanol, or 2.5 mm glucose, as indicated

in the legends to the figures. In some experiments, anti-

sequence antibodies or immunoglobulins (both at 0.25 lgIgGÆmL)1), purified from preimmune sera as described pre-

viously [34], were included in the preincubation. The pre-

incubation was stopped by removing the medium, and cells

were washed four times with NaCl ⁄Pi, at 37 �C.The uptake assay was carried out under dim light and

started by the addition of 50 nm bilirubin (7 mL) dissolved

in NaCl ⁄Pi, pH 7.4, at 37 �C. In some experiments, this

transport medium contained 100 lm taurocholate, or 2 lmdigoxin, or 1 lm nicotinic acid.

The flasks were kept in a water bath at 37 �C and gently

shaken 60 times a minute. Samples (600 lL) of the extra-

cellular solution were collected at time intervals, transferred

to conical tubes containing 600 lL methanol at 0 �C, andcentrifuged at 1200 g for 5 min. The supernatants were kept

in ice until analysis by thermal lens spectroscopy on the

same day, as described below. All experiments were per-

formed in quadruplicate.

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 lgIgGÆmL)1). Then, the medium was removed, and the cells

were washed four times with NaCl ⁄Pi, at 37 �C. The uptake

assay was started by the addition of transport medium,

consisting of either 3.5 mL 24 lm BSP or 7 mL 1.5 lmICG, in NaCl ⁄Pi, pH 7.4, at 37 �C. In some experiments,

the transport medium contained 100 lm taurocholate, or

2 lm digoxin, or 1 lm nicotinic acid, or 50 nm bilirubin.

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 analysis was performed in

batch mode on a dual-beam, 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 modula-

ted 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 (80 mm focal length) focused a helium–neon laser

(Uniphase 1103P, 632.8 nm, 2 mW; eFiberland, Fremont,

CA, USA) probe beam in front of the sample cell. The dis-

tance between the second lens and the sample cell, 138 mm,

was determined experimentally to achieve the maximal ther-

mal lens spectrometric signal. A dichroic mirror provided

collinear propagation of the pump beam and the probe

beam through the sample cell. An optical filter placed in

front of the photodiode removed the pump beam, allowing

the probe beam only to reach the photodiode. Changes in

the intensity on the probe beam axis, which are propor-

tional to the absorbance of the sample, were detected using

a silicon photodiode (Thorlabs Inc., Newton, NJ, USA;

model 201 ⁄ 5797227), situated 1.5 m beyond the sample cell.

The photodiode was connected to a lock-in amplifier (Stan-

ford Research Instruments, Sunnyvale, CA, USA; model

SR830), amplifying only the component of the input signal

that appears with the frequency of the reference signal from

the modulation. This is achieved by a Fourier transforma-

tion of the signal while filtering out all other frequencies

Fig. 11. Experimental set up for measurements with a dual-beam,

mode-mismatched thermal lens spectrometer in batch mode. Two

laser beams (pump and probe laser beams) pass through the sam-

ple cell. The pump beam induces the photothermal effect, consist-

ing of a change in the refractive index of the sample, proportional

to the concentration of the analyte. The refractive index change can

be quantified by the deflection of the probe beam. The instrumental

set up is described in Experimental procedures. This figure is not

to scale.



with a low-pass filter. The readings of the signal values

were made directly from the lock-in amplifier.

Spectrophotometric analysis

This was carried out using a UV ⁄VIS spectrophotometer

(Ultrospec 2100 pro; Amersham Biosciences, AB, Uppsala,

Sweden).

BSP

Aliquots of 100 lL of the extracellular solutions containing

BSP were added to cuvettes containing 900 lL 0.1 m

NaOH and analysed at k ¼ 580 nm (e ¼ 64 000 m)1Æcm)1).

ICG

Aliquots of 800 lL of the extracellular solutions containing

ICG were analysed at k ¼ 780 nm (e ¼ 77 500 m)1Æcm)1).

SDS ⁄PAGE and immunoblot analysis

Aliquots of the postmitochondrial fraction of HepG2 homo-

genates were resuspended in loading buffer (4 mg pro-

teinÆmL)1) and incubated at 100 �C for 5 min; the samples

(80 lg protein) were separated by SDS ⁄PAGE (12% gel).

The proteins were transferred to a nitrocellulose membrane

by electroblotting for 2 h at 2 mAÆcm)2 with the semi-dry

multiphor protein blotter system (Pharmacia Biotech,

Milano, Italy).

The membrane was kept for 2 h in blocking solution

[0.15 m NaCl, 50 mm Tris ⁄HCl, pH 7.5, containing 0.05%

(v ⁄ v) Tween 20 and 3% (w ⁄ v) skimmed milk] and then incu-

bated overnight at 4 �C with the primary antibody (antibody

A, 1.5 lg IgGÆmL)1) diluted in the blocking solution. After

being washed, the blotted membrane was incubated with an

alkaline phosphatase-conjugated secondary antibody [goat

anti-(rabbit IgG) Ig; dilution 1 : 1000; Sigma, Milan, Italy]

for 1 h at room temperature. The membrane was extensively

washed and finally stained by the addition of bromochloro-

indolyl phosphate and nitroblue tetrazolium.

Scanning electron microscopy

HepG2 cells were seeded on ThermanoxTM (Nunc, New

York, NY, USA) disks (diameter, 13 mm). The latter were

rinsed three times with NaCl ⁄Pi and fixed by treatment

with 2% (v ⁄ v) paraformaldehyde in 0.15 m cacodylate buf-

fer (pH 7.4) for 1 h at room temperature. After being

washed three times with the same buffer, disks were incuba-

ted in the presence of a blocking solution [NaCl ⁄Pi, con-

taining 1% (w ⁄ v) BSA and 20% (v ⁄ v) normal goat serum]

for 1 h at room temperature in a humidity chamber. The

blocking solution was then removed and disks were

incubated in the presence of the primary antibody solution

[NaCl ⁄Pi, containing 1% (w ⁄ v) BSA, 1% (v ⁄ v) normal goat

serum, 4% (v ⁄ v) fetal bovine serum, 0.1% (v ⁄ v) Tween 20

and antibody A at a concentration of 3.5 lg IgGÆmL)1] for

2 h at room temperature in a humidity chamber. This solu-

tion was removed and disks were washed 5 times in

NaCl ⁄Pi, and twice in a conditioning solution (50 mm

Tris ⁄HCl and 0.15 m NaCl, pH 8.4). Disks were transferred

to a humidified chamber for the incubation (for 2 h at

room temperature) with the colloidal gold (20 nm)-conju-

gated anti-rabbit IgGs (British BioCell, Cardiff, UK; dilu-

ted 1 : 100), dissolved in a solution consisting of 50 mm

Tris ⁄HCl, 0.15 m NaCl (pH 8.4), 1% (w ⁄ v) BSA, 1% (v ⁄ v)normal goat serum, 4% (v ⁄ v) fetal bovine serum and 0.1%

(v ⁄ v) Tween 20. At the end of the incubation, disks were

washed six times with 50 mm Tris ⁄HCl ⁄ 0.15 m NaCl

(pH 8.4) and three times with MilliQ grade water. To

enhance the detection of the colloidal gold particles, these

were coated with silver, using the Enhancer kit (British Bio-

Cell) for 5 min. The reaction was stopped by rinsing the

disks three times with water. Disks were dehydrated in gra-

ded ethanol solutions, treated with hexamethyldisilazane

(Sigma), which were finally removed by evaporation. The

disks were exposed to carbon vapours, to visualize the col-

loidal gold particles. The samples were observed under a

Leica Stereoscan 430i microscope (Leica Cambridge Ltd,

Cambridge, UK). Controls were carried out using immuno-

globulins purified from preimmune rabbit sera as described

previously [34] and displayed no colloidal gold-conjugated

secondary immunocomplexes.

Acknowledgements

Thanks are due to Professor G. L. Sottocasa and Pro-

fessor C. Tiribelli (University of Trieste), for useful dis-

cussions, and to Dr Marco Stebel (Animal Facility

Manager, CSPA, University of Trieste), for the immun-

ization and bleeding of rabbits. Financial support from

the University of Trieste (Fondi 60% and the Alpe-

Adria fellowship to A.M.), the Regione Friuli Venezia

Giulia (L.R. 3 ⁄98, art.16, fondo anno 2002), the Minis-

tero dell’Istruzione, Universita e Ricerca (PRIN pro-

jects 2002055532 and 2004070118), the Progetto D4

(European Social Fund, Regione Friuli Venezia Giulia

and Italian Ministry of Welfare) are acknowledged.

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