(Received for publication, February 14, 1995; and in revised form, July 18, 1995)
From the
Recently we expression cloned a rat liver organic anion
transport protein in Xenopus laevis oocytes (Jacquemin, E.,
Hagenbuch, B., Stieger, B., Wolkoff, A. W., and Meier, P. J.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 133-137). In the
present study, we have stably transfected the cDNA encoding this
protein into HeLa cells by using a vector containing a zinc-inducible
promoter. The parent cells have virtually no baseline transport of
[S]sulfobromophthalein, whereas the induced
transfected cells express a novel 74-kDa protein and avidly transport
this ligand. Transport by these cells is saturable (K
= 3.3 µM, V
= 257 pmol/min/mg protein),
bidirectional, and highly temperature-dependent. In the presence of
albumin, uptake of [
S]sulfobromophthalein
requires the presence of extracellular Cl
, whereas in
the absence of albumin, this Cl
dependence is not
seen. These studies indicate that cellular uptake of
sulfobromophthalein does not result from direct interaction with the
plasma membrane lipid bilayer but rather requires the presence of a
specific plasma membrane transporter.
Hepatocyte uptake of the organic anions bilirubin and
sulfobromophthalein (BSP) ()has kinetics suggestive of a
carrier-mediated process(1, 2) . These organic anions
circulate tightly bound to albumin from which they are rapidly
extracted by hepatocytes(2, 3) . Previous studies
performed in short term cultured rat hepatocytes demonstrated a high
affinity low capacity organic anion
transporter(4, 5) . This transporter extracted BSP
from albumin, was electroneutral and temperature-dependent, and was
inhibited after depletion of cellular ATP. The ability of this
transporter to extract BSP from albumin was modulated by extracellular
Cl
. Although a number of putative hepatocyte surface
membrane organic anion transporters have been
described(6, 7, 8) , we recently cloned a
cDNA encoding a unique rat liver protein that conferred, in Xenopus oocytes, the organic anion transport characteristics previously
described in cultured rat hepatocytes(9, 10) .
In
the present study, we have stably transfected the cDNA encoding the
organic anion transporting polypeptide (oatp) of rat liver into HeLa
cells using a vector containing a zinc-inducible promoter. The parent
cells have virtually no baseline transport of
[S]BSP, whereas the induced transfected cells
express a novel 74-kDa protein and avidly transport this ligand. This
has permitted dissection of the transport characteristics of oatp in a
mammalian system without the possible confounding effects of other cell
surface organic anion transporters.
Figure 1:
Uptake of [S]BSP
by zinc-induced control and transfected HeLa cells. Parent HeLa cells (H), HeLa cells transfected by pMEP4 vector alone (V), or 15 clones of selected HeLa cells transfected by
pMEP4-oatp were incubated for 48 h in the presence or absence of zinc,
as described under ``Materials and Methods.'' There were
approximately 500,000 cells/plate. Uptake over 15 min of 0.8 µM [
S]BSP was then determined. There was
virtually no uptake of BSP by parental- or vector-transfected cells.
Cells transfected by pMEP4-oatp transported
[
S]BSP only following zinc induction. Results
represent the means of duplicate
determinations.
Figure 2:
Time course of induction of
[S]BSP transport in pMEP4-oatp-transfected HeLa
cells by zinc. pMEP4-oatp cells were incubated in the presence of 100
µM ZnSO
(closed circles). In one set
of cells (open circle), an additional 50 µM
ZnSO
was added to the medium at 20 h. Results are expressed
as the means ± S.D. of triplicate
determinations.
Figure 3:
Immunologic detection of oatp in induced
pMEP4-oatp-transfected HeLa cells. 10% SDS-polyacrylamide gel
electrophoresis was performed on parental HeLa cells (lanes 1 and 2), pMEP4-transfected HeLa cells (lanes 3 and 4), or clone 10 pMEP4-oatp-transfected HeLa cells (lanes 5 and 6) without (lanes 1, 3, and 5) or with (lanes 2, 4, and 6) 48 h induction with ZnSO as described under
``Materials and Methods.'' Following transfer to
nitrocellulose, oatp was detected by immunoblot using a rabbit
polyclonal antibody made to a synthetic peptide corresponding to the
oatp sequence near the carboxyl terminus.
Figure 4:
Uptake of [S]BSP
over time by zinc-induced pMEP4-oatp-transfected HeLa cells.
[
S]BSP uptake by these cells at 37 °C is
rapid and increases with time. Linearity is approximated for 2-3
min. At 15 min, as much as 40-50% of incubated ligand is
cell-associated. There is little cell association of ligand at 4
°C. Results are expressed as the means ± S.D. of triplicate
determinations.
Figure 5:
Temperature dependence of
[S]BSP uptake by zinc-induced
pMEP4-oatp-transfected HeLa cells. Uptake of
[
S]BSP was determined over 15 min at
temperatures between 4 and 37 °C. Results are expressed as the
means of duplicate determinations.
Figure 6:
Effect of cellular ATP depletion on uptake
of [S]BSP by zinc-induced pMEP4-oatp-transfected
HeLa cells. When ATP was depleted by preincubation of cells in a
mixture of 0.1% sodium azide and 50 mM 2-deoxyglucose,
[
S]BSP uptake was reduced by approximately 50%.
Results are expressed as the means ±
S.D.
Figure 7:
Saturation kinetics of
[S]BSP uptake by zinc-induced
pMEP4-oatp-transfected HeLa cells. Cells were incubated in
NaCl-containing medium or in medium in which NaCl was replaced
isosmotically by sucrose. Initial uptake at 37 °C of various
concentrations of [
S]BSP was determined over 1
min, and the results were computer fit to a single class of binding
sites by a nonlinear least squares regression method. The circles represent experimental data (means of triplicate determinations),
and the lines represent the computer fit to the data. In this
representative study, in NaCl-containing medium K
was 3.2 µM and V
was 239
pmol/min/mg protein. In sucrose-substituted medium, K
was 4.4 µM and V
was 251 pmol/min/mg
protein.
Figure 8:
Influence of albumin on uptake of
[S]BSP by zinc-induced pMEP4-oatp-transfected
HeLa cells. A, [
S]BSP transport over 15
min was determined in uninduced (
) and zinc-induced (
)
pMEP-4-oatp-transfected HeLa cells and was inversely related to the
concentration of BSA in the medium. Each point represents the mean of
triplicate determinations. Studies performed at high [BSA]
are shown in the inset, in which results represent the means
± S.D. of triplicate determinations. B,
Cl
-dependent [
S]BSP uptake was
determined as the difference between uptake in NaCl-containing and
sucrose-substituted media as a function of the concentration of BSA.
Results represent the means of triplicate
determinations.
Figure 9:
[S]BSP transport by
zinc-induced pMEP4-oatp-transfected HeLa cells is bidirectional. Cells
were loaded by incubation for 15 min with 0.8 µM [
S]BSP at 37 °C. They were then washed
and incubated for various times in 5% BSA at 4 (
) or 37 °C
(
). Subsequently, cell-associated radioactivity was determined.
Results represent the means of triplicate
determinations.
Although hepatocyte transport of organic anions such as
bilirubin and BSP has been known for many years to have
carrier-mediated kinetics, characterization of this important pathway
has been difficult. In part this may be due to multiple transporters of
high and low affinities that may be present on the hepatocyte
surface(17, 18) . In previous
studies(4, 5) , we have functionally characterized
high affinity transport of [S]BSP by short term
cultured rat hepatocytes. We have found that these cells have the
ability to extract BSP from a molar excess of albumin. This uptake
process is highly temperature-dependent and is markedly reduced
following depletion of cellular ATP levels. Whether ATP interacts
directly or indirectly with this transporter is not known. Uptake of
BSP was not altered by removal of Na
from medium and
replacement with other cations such as K
,
Li
, or choline. In contrast, replacement of
Cl
in medium by gluconate or
HCO
significantly reduced BSP uptake. The
mechanism for this Cl
dependence of transport is not
known. Affinity of BSP for albumin is
Cl
-independent(5) . Although there was a
7-fold increase in affinity of hepatocytes for BSP in the presence as
compared with the absence of Cl
, it is not clear that
this is sufficient to explain the large effect on BSP transport.
Using Cl-dependent extraction of BSP from albumin
as an assay of expression, a cDNA encoding an oatp has recently been
cloned in Xenopus oocytes(9, 10, 19) . In this system,
uptake of BSP was Cl
-dependent in the presence of
albumin but Cl
-independent in its absence (10) . Because of the relatively low transport signal and the
inherent difficulties of the Xenopus oocyte system, the
present study developed and characterized HeLa cells permanently
transfected with a eukaryotic expression vector containing an inducible
oatp cDNA. These cells have virtually no baseline transport of BSP,
eliminating the possibility that an endogenous transporter could
complicate interpretation of experiments. As seen in Fig. 1, of
15 cell clones that were randomly selected, all were transporters of
BSP upon zinc induction. Transport activity declined between 20 and 48
h of induction, unless additional zinc was added to the medium (Fig. 2). The reason for this finding is not clear at this time
but likely represents depletion of the metal from the medium.
As
there was virtually no basal BSP transport in control or uninduced
transfectants, initial studies were performed in the absence of
extracellular albumin. These studies revealed rapid,
temperature-dependent uptake of BSP. This transport was not
Cl-dependent. In the presence of albumin, the rate of
BSP uptake fell considerably (Fig. 8A). However, close
to 80% of BSP transport in the presence of albumin was
Cl
-dependent (Fig. 8B). These data
thus strongly suggest that Cl
does not directly
modulate transport activity of oatp. Some studies(5, 20) have indicated that although Cl
binds to
albumin(21) , it does not influence binding of organic anions
to albumin. The mechanism resulting in Cl
-dependent
kinetics that has been described in cultured hepatocytes (4, 5, 17) and perfused rat liver (4) thus requires further clarification.
Evidence presented in this study indicates that induced transfected HeLa cells transport BSP bidirectionally. Efflux of ligand is rapid and is temperature-dependent. It is possible, however, that these cells also have an endogenous organic anion efflux mechanism that is detectable only when BSP has entered the cell. This situation has been described for efflux of taurocholate from Xenopus oocytes(22) . The fact that BSP effluxes rapidly may complicate interpretation of studies performed in the presence of extracellular albumin. This protein binds BSP avidly and markedly reduces its free concentration(5) . The fact that in the absence of albumin BSP accumulation over time reaches an apparent steady state (Fig. 4) suggests that equilibration of free BSP between the medium and the cell is established; that is, influx of ligand equals its efflux at this time. The presence of albumin outside the cell will shift the equilibrium in that direction, reducing cellular accumulation of the ligand. It should be noted that HeLa cells contain several cytosolic glutathione transferases that may bind intracellular BSP(23) . It is likely that in vivo, bile canalicular excretion provides the driving force for the net transport of BSP by keeping the system in a state of disequilibrium.
It has been suggested that uptake of the organic anion bilirubin can be explained simply by its direct interaction with the plasma membrane lipid bilayer(24) . The present studies clearly show that in the case of BSP this is not so. Parental HeLa cells have virtually no uptake of this ligand. It is not until transfected cells are induced to express oatp that they develop the ability to transport BSP (Fig. 1). Thus, oatp facilitates the movement of BSP across the lipid bilayer. The mechanism by which it does this is as yet unknown. Previous observations in the isolated perfused rat liver suggested that the uptake mechanisms for bilirubin and more soluble organic anions are partially independent(25) . Although investigations in transfected Xenopus laevis oocytes revealed inhibition of BSP uptake by bilirubin(19) , whether oatp is an efficient transporter of bilirubin has not as yet been studied directly.
These studies thus establish in a mammalian system that the presence of oatp is sufficient to permit BSP transport. The characteristics of transport resulting from oatp transfection are identical to those that have been described previously in cultured rat hepatocytes. These studies do not imply, however, that oatp is the only basolateral plasma membrane organic anion transporter in the rat hepatocyte. Several putative transporters have been suggested by a number of other investigators(6, 7, 8, 18) . Elucidation of their possible functional relationship with each other and with oatp remains to be established.