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J Biol Chem, Vol. 273, Issue 26, 16184-16191, June 26, 1998
From the oatp1 is an hepatic sinusoidal organic anion
transporter that mediates uptake of various structurally unrelated
organic compounds from blood. The driving force for uptake on oatp1 has
not been identified, although a role for bicarbonate has recently been proposed. The present study examined whether oatp1-mediated uptake is
energized by efflux (countertransport) of intracellular reduced glutathione (GSH), and whether hydrophobic glutathione
S-conjugates such as leukotriene C4
(LTC4) and S-dinitrophenyl glutathione (DNP-SG)
form a novel class of substrates for oatp1. Xenopus laevis oocytes injected with the complementary RNA for oapt1 demonstrated higher uptake of 10 nM [3H]LTC4
and 50 µM [3H]DNP-SG, and higher efflux of
[3H]GSH (2.5 mM endogenous intracellular GSH
concentration). The oatp1-stimulated LTC4 and DNP-SG uptake
was independent of the Na+ gradient,
cis-inhibited by known substrates of this transport protein
and by 1 mM GSH, and was saturable, with apparent
Km values of 0.27 ± 0.06 and 408 ± 95 µM, respectively. Uptake of [3H]taurocholate, an endogenous substrate of oatp1, was
competitively inhibited by DNP-SG. Of significance, oatp1-mediated
taurocholate and LTC4 uptake was cis-inhibited
and trans-stimulated by GSH, and [3H]GSH
efflux was enhanced in the presence of extracellular taurocholate or
sulfobromophthalein, indicating that GSH efflux down its large electrochemical gradient provides the driving force for uptake via
oatp1. The stoichiometry of GSH/taurocholate exchange was 1:1. These
findings identify a new class of substrates for oatp1 and provide
evidence for GSH-dependent oatp1-mediated substrate transport.
A major hepatic function is the clearance of a multitude of
metabolic products and foreign compounds (xenobiotics) from the blood.
Hepatic uptake of organic solutes across the sinusoidal membrane is
mediated by multiple transport systems, and four transporters have now
been cloned. Ntcp mediates Na+-coupled transport of bile
acids (1), oct1 mediates H+-coupled uptake of organic
cations (2), and oatp1 and oatp2 are related multispecific transporters
for which driving force has not yet been identified (3-5). A recent
study indicates that HCO3 oatp1-mediated transport is independent of ATP and of transmembrane
Na+, K+, or H+ gradients (3-5).
Transport is also independent of the Cl One solute that could function as an intracellular substrate for oatp1
and related organic anion transporters is reduced glutathione (GSH).
This anionic tripeptide is present in high concentrations in virtually
all cells, including hepatocytes (~10 mM), whereas plasma
concentrations are only about 10 µM (8-10). The high GSH chemical gradient coupled with the negative intracellular membrane potential could provide a substantial driving force for uptake via
oatp1.
Export of GSH into the extracellular space is the initial and perhaps
limiting step in the turnover of the tripeptide in all mammalian cells
(8-10); however, the transport system or systems that mediate GSH
efflux have not been identified. The previously reported cloning of
sinusoidal and canalicular GSH transporters has not been confirmed (14)
and appears to be an artifact (10, 14). Rates of GSH efflux differ
between cell types and are roughly equivalent to the rates of synthesis
under physiological conditions. The liver is quantitatively the major
site of synthesis in the body, and the major supplier of plasma GSH
(8-10). Studies in intact hepatocytes and isolated sinusoidal plasma
membrane vesicles indicate that GSH efflux is mediated by a low
affinity, ATP-independent, electrogenic carrier (11-14). Transport is
bidirectional, although under physiological conditions unidirectional
transport down its concentration gradient is favored, i.e.
efflux of GSH from hepatocytes (11-14).
The present study also examined whether leukotriene C4
(LTC4)1 is a
substrate for oatp1. The cysteinyl leukotrienes are potent mediators of
inflammatory responses (15), and the liver is the major site of their
removal from the blood (15-18). The mechanism for their hepatic uptake
is unknown, although evidence from isolated hepatocytes and perfused
rat liver studies indicate that uptake is mediated by oapt1 (17-19).
LTC4 uptake into isolated rat hepatocytes and plasma
membrane vesicles is independent of the Na+ gradient, is
inhibited by substrates of oatp1, and exhibits an apparent
Km of 0.20 µM (19). Once taken up into
hepatocytes, LTC4 is transported across the canalicular
membrane into bile by the ATP-dependent transporter mrp2
(which is also called cMRP or cMOAT). Hepatic uptake of another
glutathione S-conjugate (2,4-dinitrophenyl-glutathione, DNP-SG) also displays characteristics of oatp1-mediated transport (20).
To examine the hypothesis that oatp1 mediates uptake of these
glutathione S-conjugates, the present study measured
LTC4 and DNP-SG uptake in Xenopus laevis oocytes
injected with oatp1 complementary RNA (cRNA). The results demonstrate
that LTC4 and DNP-SG are substrates for this multispecific
organic solute transporter, and that hepatic uptake is energized by
countertransport of intracellular GSH.
Materials and
Animals--
[14,15,19,20-3H]Leukotriene C4
(165 Ci/mmol), [glycine-2-3H]glutathione (50 Ci/mmol), and [3H(G)]taurocholic acid (3.47 Ci/mmol) were
purchased from NEN Life Science Products. Chemicals and reagents were
obtained from Sigma, Aldrich, or J. T. Baker. Mature X. laevis were purchased from Nasco, Fort Atkinson, WI. Animals were
maintained under a constant light cycle at a room temperature of
18 °C.
Identification of Glutathione as a Driving Force and
Leukotriene C4 as a Substrate for oatp1, the Hepatic
Sinusoidal Organic Solute Transporter*
,,¶
Department of Environmental Medicine,
University of Rochester School of Medicine, Rochester, New York 14642 and the § Division of Clinical Pharmacology and Toxicology,
Department of Medicine, University Hospital,
CH-8091 Zürich, Switzerland
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
efflux may
be involved in oatp1-mediated transport (6); however, because
intracellular pH is lower than extracellular pH, the
HCO3 gradient is unlikely to energize
transport of organic anions on oatp1. Indeed, Satlin and co-workers (6)
demonstrated that oatp1-mediated cellular uptake of taurocholate was
accelerated only when the pH gradient was reversed from the normal
physiological condition. Under all other pH gradient conditions
examined by these investigators, taurocholate uptake was lower than
that seen in controls, contradicting the hypothesis that the
bicarbonate gradient energizes oatp1-mediated transport.
gradient when
measured in the absence of albumin (3). However, oatp1-mediated
transport is bidirectional (7), suggesting that solute uptake may be
energized by efflux of an intracellular solute. Whether oatp1 actually
functions as an antiporter (exchanger) and which intracellular solute
may be involved remain to be established.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Synthesis of oatp1 and Ntcp cRNA--
The cDNAs for the rat
organic anion transporting polypeptide (oatp1) and the rat
Na+/taurocholate cotransporter (Ntcp) were prepared as
described previously (1, 3). Capped cRNA was transcribed in
vitro with T3 or T7 RNA polymerase (Ambion, Austin, TX), and the
reactions were terminated by digestion of the DNA templates with
RNase-free DNase I. cRNAs were precipitated with lithium chloride and
stored at 
70 °C until used for oocyte microinjection.
Isolation of Rat Liver mRNA-- Total RNA from rat liver was prepared using the guanidinium thiocyanate/cesium chloride method (25), and mRNA was purified by chromatography with oligo(dT)-cellulose using an mRNA purification kit (Amersham Pharmacia Biotech).
Preparation and Microinjection of X. laevis Oocytes-- Isolation of X. laevis oocytes was performed as described by Goldin (26) and as described previously by our laboratory (27). Frogs were anesthetized by immersion for 15 min in ice-cold water containing 0.3% tricaine (Sigma). Oocytes were removed from the ovary and washed with Ca2+-free OR-2 solution (in mM: 82.5 NaCl, 2 KCl, 1 MgCl2, and HEPES-Tris, pH 7.5) and incubated at room temperature with gentle shaking for 90 min in OR-2 solution supplemented with 2 mg/ml collagenase (Sigma type IA). Oocytes were transferred to fresh collagenase solution after the first 45 min of incubation. Collagenase was removed by extensive washing in OR-2 solution at room temperature. Stage V and VI defolliculated oocytes were selected and incubated at 18 °C in modified Barth's solution (in mM: 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, and 20 HEPES-Tris, pH 7.5), supplemented with penicillin (100 units/ml) and streptomycin (100 µg/ml). After 3-4 h of incubation, healthy oocytes were injected with 50 nl of oatp1 cRNA (0.5-10 ng/oocyte), 50 nl of Ntcp cRNA (0.5 ng/oocyte), or 50 ng of rat liver mRNA. Control oocytes were injected with a corresponding volume of sterile H2O. Injected oocytes were cultured at 18 °C with a daily change of modified Barth's medium. Healthy oocytes with a clean brown animal half and a distinct equator line were selected for experiments.
Transport Measurements: DNP-SG and LTC4
Uptake--
Uptake studies were performed 2-3 days after
microinjection. Oocytes were pretreated with 0.5 mM
acivicin for 30 min at room temperature to inhibit 
-glutamyl
transpeptidase activity. For uptake measurement, 6-8 oocytes were
incubated at 25 °C for 1 h in 100 µl of modified Barth's
solution, in the presence of 1 µCi of [3H]DNP-SG (or
[3H]LTC4) and 0.5 mM acivicin
(27). The uptake was stopped by adding 2.5 ml of ice-cold modified
Barth's solution and oocytes washed three times each with 2.5 ml of
ice-cold modified Barth's solution. Two oocytes each were dissolved in
a polypropylene scintillation vial with 0.2 ml of 10% sodium dodecyl
sulfate, and counted in a Packard model 4530 scintillation spectrometer
after addition of 5 ml of Opti-Fluor (Packard Instruments, Downers
Grove, IL).
Taurocholate Uptake into Oocytes-- Oocytes were injected with either 0.5 ng of Ntcp cRNA or oatp1 cRNA. Na+-dependent taurocholate (10 µM) uptake was determined at 25 °C in 100 µl in modified Barth's solution, supplemented with 2.1-3.5 µCi of [3H]taurocholate. Uptake was terminated by addition of ice-cold stop solution, which was modified Barth's solution containing 1 mM unlabeled taurocholate in order to reduce unspecific binding of tracer taurocholate (28). Inhibitors were dissolved in the uptake buffer at the indicated concentrations.
GSH Efflux in Oocytes-- Oocytes injected with either water or oatp1 cRNA were reinjected with 50 nl of [3H]GSH (0.5-1 µCi/µl) and allowed to recover for 30 min in modified Barth's solution with 0.5 mM acivicin. Oocytes were washed twice with 2.5 ml of modified Barth's solution prior to efflux studies. Efflux was measured at 25 °C in 200 µl of modified Barth's solution in the presence or absence of 50 mM GSH, 50 µM taurocholate, or 1 µM BSP. Efflux was terminated at 5, 20, or 65 min by removing the medium and counting it separately from the oocytes. The 5-min values were used as a background correction and were subtracted from the 20- and 65-min values to give 15- and 60-min efflux rates.
Changing Intracellular GSH Concentrations in Oocytes-- To increase GSH, oocytes were injected with 50 nl of different GSH stock solutions (e.g. 220 mM GSH stock, for an increase of ~20 mM in the oocytes). Oocytes were incubated at 25 °C for approximately 30 min before they were used in the experiment. To decrease GSH, oocytes were incubated in modified Barth's solution containing 5 mM buthionine sulfoximine and 5 µM 1-chloro-2,4-dinitrobenzene for 18 h prior to uptake experiments. GSH content of the oocyte was measured as described previously (27).
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RESULTS |
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Introduction
Procedures
Results
Discussion
References
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X. laevis oocytes injected with the cRNA for either oatp1 or Ntcp, the Na+-taurocholate cotransporting polypeptide, showed enhanced uptake of taurocholate (Fig. 1A), confirming previous results (1, 3, 4). Oocytes injected with oatp1 cRNA also demonstrated enhanced uptake of 10 nM LTC4 and 50 µM DNP-SG, when compared with oocytes injected with water or total liver mRNA (Fig. 1B); however, the extent of stimulation of LTC4 and DNP-SG transport was lower than that of taurocholate. In contrast, oocytes injected with Ntcp-cRNA did not show enhanced transport of these glutathione S-conjugates (Fig. 1B).
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The oatp1-mediated uptake of [3H]LTC4 and
[3H]DNP-SG increased with time over a 4-h interval (Fig.
2), and was independent of extracellular
Na+ and Cl concentrations (Fig.
3). Uptake was unaffected when
Na+ in the culture medium was replaced with
choline+, or when Cl was replaced with
gluconate (Fig. 3). Isosmotic replacement of NaCl with
sucrose also failed to affect LTC4 and DNP-SG uptake. These
results with LTC4 and DNP-SG are similar to those reported
previously for taurocholate and BSP transport on oatp1 (3, 4).
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The concentration dependence of uptake was examined in oocytes
incubated with increasing concentrations of LTC4 (10 nM to 500 nM) and DNP-SG (1 µM to
1 mM), (Fig. 4). It was not
possible to examine lower concentrations of LTC4 because of
limitations imposed by the specific activity of the isotope, nor was it
feasible to examine higher LTC4 concentrations due to its
limited solubility in aqueous medium. Uptake of LTC4 and
DNP-SG was saturable, with apparent Km values of
0.27 ± 0.06 and 408 ± 95 µM (mean ± S.D.), and Vmax values of 40 ± 35 fmol·oocyte1·h1 and 4.7 ± 0.9 pmol·oocyte1·h1, respectively (Fig. 4).
This Km value for LTC4 in
oatp1-expressing oocytes is comparable to that measured in isolated rat
hepatocytes, 0.20 µM (19), consistent with a role for
oatp1 in mediating LTC4 uptake.
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Inhibition experiments revealed that BSP, taurocholate, and bilirubin
ditaurate, substrates for oatp1, are strong inhibitors of
LTC4 and DNP-SG transport in oocytes injected with oatp1
cRNA (Table I). Conversely, DNP-SG
inhibited [3H]taurocholate uptake, and the inhibition was
competitive in nature (Fig. 5). The
Km for taurocholate uptake in the absence of DNP-SG
was 38 ± 8 µM (mean ± S.D., n = 3; Fig. 5), a value comparable to that reported previously (3, 4).
The Km was increased to 88 ± 18 µM in the presence of 100 µM DNP-SG, whereas the Vmax was unchanged (3.5 ± 0.4 and 4.7 ± 1.5 pmol·oocyte1.h1,
respectively; Fig. 5), indicating competitive inhibition. Mutual inhibition of transport provides strong evidence for a shared transport
system.
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4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid, ouabain, and other glutathione S-conjugates such as BSP-SG (0.5 mM) and S-(p-chlorophenacyl)-glutathione (1 mM) also inhibited uptake of LTC4 and DNP-SG (Table I). LTC4 uptake was decreased to 43% and 23% of control in the presence of BSP-SG and S-(p-chlorophenacyl)-glutathione, respectively. DNP-SG uptake was also inhibited by these glutathione S-conjugates, but the extent of inhibition was lower (Table I). These data indicate that bulky glutathione S-conjugates may be substrates for oatp1.
Of significance, 1 mM GSH cis-inhibited LTC4 and DNP-SG uptake, suggesting that GSH itself may be a substrate for oatp1 (Table I). Extracellular GSH (1 mM) also inhibited 1 µM [3H]taurocholate uptake by 36 ± 16% (mean ± S.D.; n = 6). However, GSSG (1 mM) did not have any significant effect (Table I).
To test the possibility that GSH is a substrate for oatp1, [3H]GSH efflux was measured in oocytes expressing oatp1. The endogenous GSH transport rate in oocytes was high (Table II), as reported previously (27), but was even higher in oatp1-expressing oocytes (Table II), consistent with the hypothesis that GSH is transported on oatp1. The presence of 50 mM unlabeled GSH in the efflux medium further accelerated [3H]GSH efflux in both control and oatp1-expressing oocytes (Table II).
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If [3H]GSH efflux is linked to oatp1-mediated uptake of organic solutes, inclusion of oatp1 substrates in the culture medium should accelerate [3H]GSH efflux. Indeed, Table II shows that inclusion of 50 µM taurocholate or 1 µM BSP in the culture medium stimulated [3H]GSH release in oatp1-expressing oocytes, but not in water-injected controls. After correction for the amount of [3H]GSH released by water-injected oocytes, a total of 0.27 ± 0.04% of intracellular GSH was released during a 15-min interval by oocytes incubated in modified Barth's medium. This value was higher in the presence of taurocholate or BSP (0.46 ± 0.10% and 0.43 ± 0.07%, respectively; Table II), consistent with GSH/organic anion exchange on oatp1.
To estimate the stoichiometry of GSH/organic anion exchange on oatp1,
the amount of taurocholate-stimulated [3H]GSH efflux
(Table II) was compared with the amount of
[3H]taurocholate taken up under similar conditions.
Measurements of [3H]taurocholate uptake were carried out
in parallel with the studies illustrated in Table II, using similar
batches of oocytes that were incubated under nearly identical
conditions. As shown in Table II, inclusion of 50 µM
unlabeled taurocholate in the incubation medium stimulated the release
of an additional 0.19% of intracellular [3H]GSH
(i.e. 0.46-0.27 = 0.19%), or approximately 2.4 pmol·oocyte1.15 min1 [(0.19%/15 min)
(0.5 µl/oocyte) (2.5 mM GSH)]. This value is nearly
identical to the amount of [3H]taurocholate taken up
during the same time interval, 2.5 ± 0.6 pmol·oocyte1.15 min1, indicating a 1:1
exchange on oatp1.
Additional evidence for GSH/organic anion exchange on oatp1 was provided by studies that measured taurocholate and LTC4 uptake in oocytes with differing intracellular GSH concentrations (Fig. 6). Taurocholate uptake in oocytes with low (~0.3 mM) intracellular GSH was 46% of control oocytes (~2.5 mM endogenous GSH; Fig. 6A). When the intracellular GSH concentration was raised to ~20 mM there was a significant trans-stimulation of taurocholate uptake to 155% of control values, suggesting that GSH is a driving force for oatp1. In contrast, oatp1-expressing oocytes loaded with 20 mM amounts of either N-acetylcysteine, L-glutamate, or glutarate did not demonstrate enhanced uptake of [3H]taurocholate (Fig. 6A), indicating that trans-stimulation is selective for GSH. Uptake of LTC4 in oatp1-expressing oocytes was also inhibited by GSH depletion, and trans-stimulated by high intracellular GSH (Fig. 6B).
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The relation between oatp1-mediated taurocholate uptake and intracellular GSH concentration is illustrated in Fig. 7. Taurocholate uptake reached a maximum at about 10 mM GSH and was not further accelerated at higher intracellular GSH concentrations, indicating a saturable transport mechanism. If one assumes a one-for-one exchange of extracellular taurocholate for intracellular GSH and Michaelis-Menten kinetics, these data indicate that the apparent Km for GSH is approximately 3 mM; however, this is only an approximation because of the indirect nature of the measurement. Given that intracellular GSH concentrations in hepatocytes are ~10 mM, the transporter should always be saturated under physiological conditions. However, GSH may become limiting for oatp1 function under conditions of extensive GSH depletion.
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To further define the role of oatp1 in GSH efflux, additional studies examined taurocholate uptake in the presence of 1 mM methionine, cystathionine, or dithiothreitol, compounds that are known to alter sinusoidal GSH release in mammalian hepatocytes (13, 29-32). In intact hepatocytes, methionine and cystathionine are effective trans-inhibitors of sinusoidal GSH efflux, and dithiothreitol a trans-stimulator of GSH efflux (13, 29-32). However, these agents had no effect on oatp1-mediated taurocholate uptake (data not shown), indicating that oatp1 is not sensitive to these agents and therefore may not be the major mechanism of GSH release from hepatocytes.
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DISCUSSION |
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Abstract
Introduction
Procedures
Results
Discussion
References
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The present study demonstrates that LTC4 and DNP-SG
are substrates for the hepatic sinusoidal organic solute transporter
oatp1, substantiating previous findings in rat hepatocytes and isolated liver plasma membrane vesicles (18-20). Uptake of these glutathione S-conjugates into oatp1-expressing oocytes was saturable,
inhibited by known substrates of oatp1, and was independent of the
Na+ and Cl gradients. The apparent
Km for LTC4 uptake in oatp1-expressing oocytes, 0.27 µM (Fig. 4), is similar to that measured in
hepatocytes, 0.20 µM (19), consistent with hepatic uptake
on oatp1. The transport characteristics of DNP-SG in oocytes expressing
oatp1 are also similar to those reported in perfused rat liver; uptake
was comparatively low, Na+-independent, and inhibited by
substrates for oatp1 (20). LTC4 and DNP-SG may of course
also be substrates for additional hepatic solute transporters,
including other members of the oatp family, and this will need to be
evaluated.
The present findings with LTC4 and DNP-SG differ from
previous studies with oatp1 (3, 4), which failed to find an effect of
LTC4 and DNP-SG on [35S]BSP uptake in
oatp1-expressing oocytes. The reason for this apparent discrepancy is
most likely related to the kinetics of transport of BSP
versus the glutathione S-conjugates on oatp1. BSP
is one of the best substrate for this transporter, with a Km of 1.5 µM, whereas DNP-SG has both
a lower affinity (Km of 408 µM) and
transport capacity (Vmax of 4.7 pmol·oocyte1.h1 for DNP-SG (Fig. 4B),
versus 9 pmol·oocyte1.h1 for
BSP (3)). Thus, high concentrations of DNP-SG are needed to elicit
significant inhibition of BSP uptake, but these concentrations were not
used in previous studies. In contrast, LTC4 has a higher affinity (Km of 0.27 µM), but its use
as an inhibitor is limited by its very low water solubility. Maximum
LTC4 concentrations in aqueous medium are on the order of
0.5 µM, a concentration that is expected to have minimal
effects on BSP uptake via oatp1.
Our results also provide direct evidence that intracellular GSH is a driving force for solute uptake on oatp1. First, the observation that glutathione S-conjugates are substrates for oatp1 indicates that the GSH moiety may be a determining factor in substrate recognition. Second, GSH cis-inhibited LTC4, DNP-SG, and taurocholate uptake, indicating a shared transport mechanism. Third, oatp1-mediated taurocholate uptake was trans-stimulated by GSH, but not by glutarate, L-glutamate, or N-acetylcysteine, providing strong evidence for GSH/taurocholate exchange. The relation between intracellular GSH and oatp1-mediated taurocholate uptake (Fig. 7), indicates that transport is maximal at GSH concentrations that are comparable to those of intact hepatocytes, ~10 mM, and gradually declines at lower GSH concentrations. Thus, oatp1 function may be compromised by GSH availability if GSH levels are significantly decreased. Fourth, [3H]GSH efflux was higher in oatp1-expressing oocytes, and was further accelerated in the presence of extracellular taurocholate, BSP, and high concentrations of GSH, providing strong evidence for GSH/organic anion exchange on oatp1. Fifth, a comparison of the amount of GSH released and taurocholate taken up on oatp1 indicates one-for-one exchange of these monovalent anions.
Additional evidence for GSH/organic solute exchange on oatp1 comes from previous studies of GSH transport in rat liver sinusoidal membrane vesicles (11). GSH transport in sinusoidal vesicles was cis-inhibited and trans-stimulated by BSP-SG, a substrate for oatp1, suggesting that GSH efflux may drive the uptake of organic anions into hepatocytes (11). Studies with canalicular membrane vesicles have also demonstrated trans-stimulation between GSH and certain organic solutes (33, 34), suggesting that GSH/organic solute exchange may be a general mechanism.
To date, two major families of ATP-independent organic anion
transporters have been identified. One family comprises the organic anion/dicarboxylate exchangers, and includes kidney oat1 (or roat1) (35-37). Cellular uptake of organic anions on kidney oat1 was recently demonstrated to be directly coupled to efflux of 
-ketoglutarate (35,
36). The second family comprises the oatp-related transporters, and
includes liver oatp1 and oatp2 (3-5), kidney-specific oat-k1 (38), and
the prostaglandin transporter pgt (39). The transporters in this family
exhibit approximately 30-80% amino acid identity (40). The driving
force for uptake on the oatp-related transporters has not been
identified.
The present findings with oatp1 raise the possibility that GSH exchange may be a common mechanism for this family of transporters. However, it is important to note that, despite the high sequence homology among the oatp-related transporters, there are major differences in substrate specificity and possibly in energy coupling. For example, oatp1 and oat-k1 share 72% amino acid identity, yet oat-k1 is unable to transport either taurocholate or LTC4 (38), whereas oatp1 accepts both as substrates (Ref. 3 and present study). Additional studies are needed to characterize the substrate specificity and driving forces for the oatp-related transporters.
The overall contribution of oatp1 to sinusoidal GSH efflux is unknown, but it is unlikely to be the predominant mechanism of GSH release in hepatocytes. Studies characterizing sinusoidal GSH efflux reveal that methionine and cystathionine trans-inhibit GSH efflux in hepatocytes (29, 30, 32), and dithiothreitol trans-stimulates GSH efflux (13, 31). In the present study, methionine, cystathionine, and dithiothreitol did not affect oatp1-mediated taurocholate transport, indicating that this putative GSH efflux mechanism is not altered. Taken together, these findings are consistent with the presence of at least two GSH transport systems in the sinusoidal membrane (Fig. 8): a putative GSH transporter (gsht) that is sensitive to methionine, cystathionine, and dithiothreitol; and oatp1, which is unaffected by these compounds. trans-Stimulation of GSH efflux by BSP or BSP-SG may occur through oatp1, whereas cis-inhibition may occur through both systems. It is plausible that other oatp1-like transporters that have recently been cloned may also be contributing to GSH efflux (5), and this possibility is currently being examined in our laboratory.
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Previous studies have shown that sinusoidal GSH release in perfused rat liver is inhibited by BSP, rose bengal, indocyanine green, and bilirubin (41), substrates of oatp1. Inhibition occurs from an intracellular site and is reversible (41); inhibition is overcome when these anions are cleared from the cell by transport across the canalicular membrane into bile. This cis-inhibition of the putative sinusoidal GSH transporter by some oatp1 substrates may be of physiologic significance in that it would maximize intracellular GSH concentrations for organic anion uptake on oatp1 (Fig. 8). Interestingly, transport of these same organic anions into bile is mediated by mrp2, a transporter that also appears to function optimally when intracellular GSH concentrations are high (42-44). Thus, the inhibition of sinusoidal GSH efflux by oatp1 substrates, and the resulting higher cellular GSH concentrations, may accelerate both hepatic uptake and biliary excretion of these compounds. The net effect is faster hepatobiliary clearance of chemicals, many of which are potentially toxic (Fig. 8).
In conclusion, the present study demonstrates that GSH is a driving force for oatp1 and that glutathione S-conjugates are a novel class of substrates for this transporter. oatp1 may therefore contribute to sinusoidal GSH release and the regulation of GSH turnover in hepatocytes. The data are consistent with the presence of multiple GSH efflux mechanisms, and explain several observations regarding GSH efflux characteristics in intact hepatocytes. However, confirmation of this model awaits the molecular characterization of additional GSH transport proteins.
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FOOTNOTES |
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* This work was supported in part by National Institute of Health Grants DK48823 and ES06484, NIEHS Center Grant ES01247, Toxicology Training Program Grant ES07026, and Swiss National Science Foundation Grant 31-45536.95.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 716-275-0262; Fax: 716-256-2591; E-mail: ballatorin{at}envmed.rochester.edu.
1 The abbreviations used are: LTC4, leukotriene C4; BSP, bromosulfophthalein; BSP-SG, glutathione S-conjugate of BSP; DNP-SG, S-(2,4-dinitrophenyl)-glutathione; cRNA, complementary RNA.
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REFERENCES |
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Abstract
Introduction
Procedures
Results
Discussion
References
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B. S. Chan, J. A. Satriano, and V. L. Schuster Mapping the Substrate Binding Site of the Prostaglandin Transporter PGT by Cysteine Scanning Mutagenesis J. Biol. Chem., September 3, 1999; 274(36): 25564 - 25570. [Abstract] [Full Text] [PDF]
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J.-i. Nishino, H. Suzuki, D. Sugiyama, T. Kitazawa, K. Ito, M. Hanano, and Y. Sugiyama Transepithelial Transport of Organic Anions across the Choroid Plexus: Possible Involvement of Organic Anion Transporter and Multidrug Resistance-Associated Protein J. Pharmacol. Exp. Ther., July 1, 1999; 290(1): 289 - 294. [Abstract] [Full Text]
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U. Eckhardt, A. Schroeder, B. Stieger, M. Hochli, L. Landmann, R. Tynes, P. J. Meier, and B. Hagenbuch Polyspecific substrate uptake by the hepatic organic anion transporter Oatp1 in stably transfected CHO cells Am J Physiol Gastrointest Liver Physiol, April 1, 1999; 276(4): G1037 - G1042. [Abstract] [Full Text] [PDF]
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E. Tan, R. G. Tirona, and K. S. Pang Lack of Zonal Uptake of Estrone Sulfate in Enriched Periportal and Perivenous Isolated Rat Hepatocytes Drug Metab. Dispos., March 1, 1999; 27(3): 336 - 341. [Abstract] [Full Text]
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 M. Sjölinder, S. Tornhamre, H.-E. Claesson, J. Hydman, and J. A. Lindgren Characterization of a leukotriene C4 export mechanism in human platelets: possible involvement of multidrug resistance-associated protein 1 J. Lipid Res., March 1, 1999; 40(3): 439 - 446. [Abstract] [Full Text]
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J. F. Rebbeor, G. C. Connolly, M. E. Dumont, and N. Ballatori ATP-dependent Transport of Reduced Glutathione on YCF1, the Yeast Orthologue of Mammalian Multidrug Resistance Associated Proteins J. Biol. Chem., December 11, 1998; 273(50): 33449 - 33454. [Abstract] [Full Text] [PDF]
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E. Battaglia and J. Gollan A Unique Multifunctional Transporter Translocates Estradiol-17beta -Glucuronide in Rat Liver Microsomal Vesicles J. Biol. Chem., June 22, 2001; 276(26): 23492 - 23498. [Abstract] [Full Text] [PDF]
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W. Wang, D. J. Seward, L. Li, J. L. Boyer, and N. Ballatori Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate PNAS, July 31, 2001; 98(16): 9431 - 9436. [Abstract] [Full Text] [PDF]
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