(Received for publication, August 25, 1994; and in revised form, December 1, 1994)
From the
A large number of structurally distinct electrophiles are
conjugated to glutathione within hepatocytes, and the resulting
glutathione S-conjugates are selectively transported across
the canalicular membrane into bile. To test the hypothesis that a
single multi-specific, ATP-dependent carrier mediates biliary secretion
of glutathione S-conjugates, the present study compared the
driving forces and substrate specificity for canalicular transport of S-ethylglutathione (ethyl-SG), a low molecular weight and
relatively hydrophilic thioether, and S-(2,4-dinitrophenyl)-glutathione (DNP-SG), a larger and more
hydrophobic anion, using isolated rat liver canalicular membrane
vesicles. In agreement with previous findings, DNP-SG transport was
stimulated by ATP, although there was considerable transport in the
absence of ATP. ATP-independent DNP-SG transport was unaffected by a
Na gradient, was enhanced by a valinomycin-induced
K
diffusion potential, and was saturable, with both
high affinity (K
= 8 ± 2
µM) and low affinity (K
= 0.5 ± 0.1 mM) components. High
affinity ATP-independent DNP-SG uptake was cis-inhibited by
GSH, GSH monoethyl ester, glutathione S-conjugates, other
-glutamyl compounds, sulfobromophthalein, and
4,4`-diisothiocyanatostilbene-2,2`-disulfonic acid (DIDS). In contrast,
ATP-dependent DNP-SG uptake was unaffected by GSH, GSH ester, S-methyl glutathione, or S-carbamidomethyl
glutathione, but was strongly inhibited by sulfobromophthalein, DIDS,
and by high molecular weight and relatively hydrophobic glutathione S-conjugates. Transport of the low molecular weight ethyl-SG
conjugate was only minimally stimulated by ATP (10-20%).
ATP-independent ethyl-SG uptake was electrogenic, saturable (K
= 10 ± 1
µM) and was inhibited by GSH and all glutathione S-conjugates tested. These findings indicate the presence of
multiple canalicular transport mechanisms for glutathione S-conjugates and demonstrate that the physicochemical
properties of the S moiety are major determinants of
transport. Relatively high molecular weight hydrophobic conjugates are
substrates for both ATP-dependent and -independent mechanisms, whereas
low molecular weight glutathione S-conjugates are transported
largely by electrogenic carriers.
Extrusion of cellular metabolites and xenobiotics from the hepatocyte across the canalicular plasma membrane into bile is mediated in part by at least three distinct ATP-dependent transport systems; the P-glycoproteins (multidrug resistance proteins), an export pump for monovalent conjugated bile acids, and a transporter for non-bile acid organic anions (reviewed by Arias et al., 1993; Meier and Steiger, 1993; Vore, 1993; Zimniak and Awasthi, 1993). An ATP-independent canalicular organic anion transporter has also been identified (Arias et al., 1993). These proteins display remarkably broad substrate specificities and together mediate the hepatocellular efflux of a variety of endogenous compounds and innumerable xenobiotics. Although the P-glycoproteins are well characterized at the biochemical and molecular level, the other transport mechanisms have only been examined in isolated membrane vesicle systems.
An important group of substrates for the
ATP-dependent organic anion carrier is the glutathione S-conjugates, which are formed intracellularly by the reaction
of GSH with both endogenous and exogenous electrophilic compounds
(Boyland and Chasseaud, 1969; Chasseaud, 1979; Anderson and Meister,
1983). Studies with liver canalicular membrane vesicles (Ishikawa et al., 1989, 1990; Kobayashi et al., 1990, 1991;
Akerboom et al., 1991; Nishida et al., 1992;
Fernandez-Checa et al., 1992), as well as membrane
preparations from red blood cells (Kondo et al., 1982; LaBelle et al., 1986a, 1986b), heart (Ishikawa, 1989; Ishikawa et
al., 1989), and other tissues (Schaub et al., 1991;
Ishikawa and Ali-Osman, 1993), have established that glutathione
disulfide (GSSG), and glutathione S-conjugates of
sulfobromophthalein (BSP()-SG), 1-chloro-2,4-dinitrobenzene
(DNP-SG), and a leukotriene (LTC
) are substrates for
ATP-dependent carriers. Kinetic analysis of ATP-dependent DNP-SG
transport in canalicular liver plasma membrane (cLPM) vesicles
indicates a single high affinity component of transport, with a K
ranging from 4 to 71 µM (Kobayashi et al., 1990; Akerboom et al., 1991),
whereas erythrocytes have two ATP-dependent components with K
values ranging from
1 to 8
µM and 100 to 900 µM, respectively (Bartosz et al., 1993; Kondo et al., 1982). Erythrocytes may
also have separate ATP-dependent transporters for GSSG and DNP-SG
(LaBelle et al., 1986b; Board et al., 1992), but this
has not yet been firmly established. ATP-dependent organic anion
transport has a broad substrate specificity, accepting a variety of
anionic compounds that bear little structural similarity with each
other or with the glutathione S-conjugate substrates
(Kobayashi et al., 1990; Akerboom et al. 1991; Oude
Elferink et al., 1991; Nishida et al., 1992a, 1992b;
Bartosz et al., 1993). The mechanism by which these chemically
diverse substrates are recognized by the transporter(s) is not known.
The overall functional characteristics of the ATP-dependent organic
anion transporters are comparable to those of the P-glycoproteins,
suggesting similar structural motifs and a common evolutionary origin.
Because glutathione S-conjugates are good substrates for
the ATP-dependent organic anion carrier, this transporter was named the
GS-X pump by Ishikawa(1992). However, our recent finding that
canalicular transport of a low molecular weight glutathione mercaptide
(CHHg-SG) is independent of ATP indicates that not all
glutathione S-conjugates are substrates for this GS-X pump and
suggest that the physicochemical properties of the S moiety
may determine whether the conjugate is transported on the ATP-dependent
system (Dutczak and Ballatori, 1994). Furthermore, these data indicate
the presence of at least two ATP-independent glutathione S-conjugate transport mechanisms. Studies of GSH transport in
canalicular membrane vesicles also suggest the presence of a high
affinity ATP-independent GSH transport system whose primary function
may be to transport some glutathione S-conjugates,
-glutamyl compounds, and other anions into bile (Ballatori and
Dutczak, 1994). To test these hypotheses directly, the present study
compared the driving forces and substrate specificity for canalicular
transport of two glutathione S-conjugates that differ in their
physicochemical properties, S-ethylglutathione (ethyl-SG) and S-(2,4-dinitrophenyl)glutathione (DNP-SG). Our findings
identify and characterize both ATP-dependent and -independent
glutathione S-conjugate transport mechanisms.
DNP-SG (Hinchman et
al., 1991; Saunders, 1934), BSP-SG (Whelan et al., 1970),
and S-ethacrynic acid glutathione (Ploemen et al.,
1990) were synthesized and purified as described previously. BSP-SG was
further purified to remove small amounts of contaminating BSP, GSSG,
and GSH by elution through a Sephadex G-50-fine column (1.5 30
cm). The Sephadex G-50 column was eluted with 100 mM Tris-HCl,
pH 7.5, at a flow rate of 43 ml/h at room temperature. BSP-SG
concentration was assessed by measuring absorbance at 580 nm in a
solution containing 100 mM sodium pyrophosphate buffer, pH
8.4. Glutathione monoethyl ester was synthesized based on the method of
Anderson et al.(1985) and characterized as described
(Ballatori and Truong, 1992). S-Benzyl glutathione was
synthesized from GSH and benzyl chloride by the method of Vince et
al.(1971). The product was recrystallized in water.
S-Carboxymethyl glutathione was synthesized by dissolving
equimolar amounts (1 mmol) of iodoacetate and GSH in water and
immediately adding a solution of 2 M KOH containing 3.3 M KHCO to raise the pH to 8.5. The reaction mixture was
incubated in the dark at room temperature with continuous stirring for
15 min. The product was purified by ion exchange chromatography (Fig. 1B) and detected and quantitated by reaction with
0.2% ninhydrin in 99% ethanol, at a wavelength of 570 nm (Moore and
Stein, 1948). DEAE-Sephadex A-25 column chromatography was carried out
at a flow rate of 62 ml/h, using 50 mM potassium phosphate
buffer, pH 7.5 (Fig. 1B). S-Carbamidomethyl
glutathione was synthesized from GSH and iodoacetamide using a similar
approach. Iodoacetamide and GSH (1 mmol each) were dissolved in of
water, the pH was raised to 8.0 with saturated KHCO
, and
the solution incubated in the dark at room temperature, with continuous
stirring for 1 h. The product was applied to a DEAE-Sephadex A-25
column and eluted with 10 mM potassium phosphate buffer, pH
7.5. S-Carbamidomethyl glutathione eluted in fractions
19-23 (using conditions as described in Fig. 1A)
and was quantitated by reaction with ninhydrin. GSH and GSSG eluting
from the column were detected by the method of Griffith(1980).
Figure 1:
Separation of glutathione and
glutathione S-conjugates by ion exchange chromatography.
Approximately 5 µmol of the indicated compounds were applied to a
DEAE-Sephadex A-25 column (1.5 30 cm), and eluted at room
temperature with either 10 mM (A) or 50 mM (B) potassium phosphate buffer, pH 7.5, at a flow rate of
62 ml/h. Fractions of 3 ml were collected and analyzed by reaction with
ninhydrin (as illustrated), but also by enzymatic analysis for GSH and
GSSG (Griffith, 1980) and direct absorbance at 365 nm for
DNP-SG.
[H]DNP-SG was synthesized using a similar
approach. [
H]GSH (6 nmol) was reacted with
1-chloro-2,4-dinitrobenzene (25 nmol) at 37 °C for 20 min.
1-Chloro-2,4-dinitrobenzene was recrystallized from ethanol and water
(3:2 v/v) prior to use. [
H]DNP-SG was purified
from residual [
H]GSH and
[
H]GSSG as illustrated in Fig. 1B. Radiolabeled compounds were added to the
membrane incubation solutions at a concentration of 2.5 µCi/ml,
such that the final concentration of potassium phosphate was less than
5 mM.
In some experiments, the membrane potential was clamped using the potassium ionophore valinomycin (10 µg/mg protein) in the presence of 20 mM KCl in the intra- and extravesicular spaces. Valinomycin was dissolved in dimethyl sulfoxide: the final concentration of dimethyl sulfoxide was less than 0.1%, a concentration that had no effect on transport (data not shown). Some incubation solutions were supplemented with dithiothreitol (5 mM) to ensure that sulfhydryl-containing compounds remained in their reduced forms. The corresponding controls also received 5 mM dithiothreitol. The pH of incubation solutions containing high concentrations of glutathione S-conjugates, GSH, or other anions was routinely adjusted to 7.5 immediately before use with either 1 M KOH or Tris base. Specific details regarding the composition of the vesicle incubation media are provided in the individual figure and table legends.
Uptake of radiolabeled substrates into cLPM vesicles was
measured by a rapid Millipore filtration technique (Ballatori et
al., 1986; Meier et al., 1984b). Membrane suspensions
(80-130 µg of protein in 20 µl) were preincubated at 25
or 37 °C for 15 min. Uptake studies were initiated by the addition
of 80 µl of incubation medium, also prewarmed, containing various
concentrations of the radiolabeled substrates. Transport was terminated
by the addition of 1 ml of ice-cold stop solution and immediate rapid
filtration through a 0.45-µm filter (Millipore, HAWP) that had been
premoistened with ice-cold stop solution. Vesicles were then washed
with an additional 4 ml of stop solution. Unless otherwise indicated
the stop solution consisted of 300 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 0.2 mM CaCl, and 20
mM KCl. Filters were dissolved in 5 ml of Opti-Fluor (Packard
Instrument Co., Downers Grove, IL) and counted in a Packard Tri Carb
Scintillation Counter (model 4530). Nonspecific binding of isotope to
filters and vesicles was determined in each experiment by the rapid
addition of ice-cold incubation solution, followed by cold stop
solution, to 20 µl of membrane vesicle suspension also kept at
0-4 °C. This blank was subtracted from all determinations.
All incubations were performed in triplicate and all observations
confirmed with three or more separate membrane preparations. Kinetic
parameters were calculated from the Eadie-Hofstee transformation of the
data by the method of residuals (Gibaldi and Perrier, 1975).
The
effect of ATP on [H]ethyl-SG,
[
H]DNP-SG, and
[
H]taurocholate uptake by cLPM vesicles was
measured at 37 °C in an incubation solution containing 290 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 20 mM KCl,
0.2 mM CaCl
, 10 mM MgCl
, 10
mM phosphocreatine, 100 µg/ml of creatine phosphokinase,
and either 1 mM ATP (disodium salt) or 2 mM NaCl.
Figure 2:
Effect of ATP on ethyl-SG, DNP-SG, and
taurocholate uptake in cLPM vesicles. Vesicles containing 250 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 20 mM KCl,
and 0.2 mM CaCl were pretreated with 0.25 mM acivicin. Uptake of 10 µM [
H]ethyl-SG, 10 µM [
H]DNP-SG, and of 1 µM [
H]taurocholate was measured at 37 °C in
an incubation solution containing 290 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 20 mM KCl, 0.2 mM CaCl
, 10 mM MgCl
, 10 mM phosphocreatine, 100 µg/ml creatine phosphokinase, and either
1 mM ATP (disodium salt) or 2 mM NaCl. The stop
solution contained 300 mM sucrose, 10 mM HEPES-Tris,
pH 7.5, 20 mM KCl, and 0.2 mM CaCl
. Data
are means ± S.E. of three experiments, each performed in
triplicate.
ATP-independent ethyl-SG and DNP-SG uptake occurred into an osmotically sensitive space (Fig. 3). Uptake of both solutes was linearly decreased as vesicle size diminished at higher osmolarities. The data for DNP-SG are comparable to those presented by Inoue et al.(1984), whereas those for ethyl-SG are similar to those for high affinity GSH uptake by the cLPM vesicles (Ballatori and Dutczak, 1994) and indicate a relatively small degree of binding to the vesicles. The apparent intravesicular volumes calculated from these uptake values are approximately 2.5-3.5 µl/mg of protein, providing further evidence for low nonspecific binding to the canalicular membranes.
Figure 3:
Transport of ethyl-SG and DNP-SG into an
osmotically active intravesicular space. Canalicular vesicles were
resuspended in media containing 10 mM HEPES-Tris, pH 7.5, 250
mM sucrose, and 0.25 mM acivicin. They were then
incubated at 25 °C for 30 min in media containing 10 mM HEPES-Tris, pH 7.5, 10 µM of either
[H]ethyl-SG (circles) or
[
H]DNP-SG (triangles), and from
0.3-0.7 M sucrose. Membrane blanks were performed at
each sucrose concentration. Data are means ± S.E. (n = 3).
Figure 4:
Time course of ethyl-SG and DNP-SG uptake
into cLPM vesicles in the presence of various inwardly directed cation
gradients. Canalicular membrane vesicles were resuspended in 250 mM sucrose, 10 mM HEPES-Tris, pH 7.5, and 0.25 mM acivicin. Uptake of 10 µM [H]ethyl-SG or 10 µM [
H]DNP-SG was measured at 25 °C in an
incubation solution containing 170 mM sucrose, 10 mM HEPES-Tris, pH 7.5, 10 µM [
H]ethyl-SG or 10 µM [
H]DNP-SG, and 100 mM of either
NaCl, KCl, or LiCl. Insets illustrate uptake at short time
intervals. Values are means ± S.E. of three experiments, each
performed in triplicate.
Figure 5:
Concentration dependence of initial rates
of DNP-SG uptake in cLPM vesicles in the absence of ATP. Vesicles
containing 250 mM sucrose, 10 mM HEPES-Tris, pH 7.5,
20 mM KCl, and 0.2 mM CaCl were
pretreated with 0.25 mM acivicin and 10 µg valinomycin/mg
protein. They were then incubated at 25 °C in the same media
supplemented with [
H]DNP-SG (1 µM to
6 mM), and uptake was measured after 10 s. The dashed
lines represents the nonsaturable component of uptake, obtained by
least-square linear regression analysis of the data at high
extravesicular substrate concentrations (
2 mM). The slopes
of the dashed lines in panels A and B are
similar. Inset shows the Eadie-Hofstee plot of these data over
the entire concentration range. Values are means ± S.E. of three
experiments, each performed in triplicate.
Figure 6:
Concentration dependence of initial rates
of ethyl-SG uptake in cLPM vesicles. Explanatory information is similar
to that of Fig. 5legend. Uptake of
[H]ethyl-SG (1 µM to 10 mM)
was measured after 10 s. Values are means ± S.E. of three
experiments, each performed in triplicate.
Initial rates of DNP-SG uptake were a nonlinear function
of concentration (Fig. 5). When corrected for the nonsaturable
component (dashed lines) data were obtained that follow
Michaelis-Menten kinetics. An Eadie-Hofstee plot of all of the data (Fig. 5, inset) revealed the presence of at least two
saturable transport components: a high affinity system with an apparent K of 8 ± 2 µM and a V
of 33 ± 6
pmol
mg
10 s
, and a low
affinity system with a K
of 0.5 ± 0.1
mM and a V
of 526 ± 75
pmol
mg
10 s
.
A
similar kinetic analysis for ethyl-SG (Fig. 6) revealed the
presence of a single high affinity saturable component, with an
apparent K of 10 ± 1 µM and a V
of 6 ± 2
pmol
mg
10 s
. At
extravesicular ethyl-SG concentrations up to 10 mM, there was
no evidence for a second low affinity system (Fig. 6B).
ATP-dependent DNP-SG transport was unaffected by GSH, GSH monoethyl ester, or by low molecular weight and relatively hydrophilic glutathione S-conjugates, such as S-methyl glutathione, S-carbamidomethyl glutathione, or glutathionesulfonic acid (Table 2). In contrast, larger and more hydrophobic glutathione S-conjugates were powerful inhibitors. ATP-dependent transport was essentially completely inhibited by 1 mM DNP-SG, BSP-SG, GSSG, S-octyl, S-(p-nitrobenzyl), S-(p-chlorophenacyl), and S-ethacrynic acid glutathione. For the S-alkyl conjugates, the extent of inhibition increased as the chain length increased from methyl to octyl (Table 2), confirming previous findings (Kobayashi et al., 1990; Ishikawa et al., 1990), and indicating that hydrophobicity is an important determinant of ATP-dependent glutathione S-conjugate transport.
A comparison of the effects of S-carboxymethyl glutathione (GS-CH-COOH) to those
of S-carbamidomethyl glutathione
(GS-CH
-CO-NH
) indicates that charge may also be
an important determinant of transport on the ATP-dependent system. The
dianionic S-carboxymethyl conjugate decreased uptake to 69% of
control, whereas S-carbamidomethyl glutathione had no effect (Table 2), even though these compounds have similar molecular
weights and hydrophobicity.
ATP-dependent DNP-SG uptake (5
µM) was also nearly completely inhibited by BSP (100
µM) and DIDS (100 µM) and significantly
decreased by taurocholate (250 µM), but was unaffected by
other anionic compounds including the GSH analogs -Glu-AIB-Gly and
-Glu-Gly-Gly, the dipeptides
-Glu-Cys and Cys-Gly, and the
amino acids glutamate and cysteine (Table 2). The inhibition by
BSP, DIDS, and taurocholate supports previous studies indicating that
the ATP-dependent carrier can transport a variety of organic anions, in
addition to glutathione S-conjugates (Ishikawa et
al., 1989, 1990; Kobayashi et al., 1990, 1991; Akerboom et al., 1991; Oude Elferink et al., 1991; Nishida et al., 1992; Fernandez-Checa et al., 1992; Bartosz et al., 1993).
In contrast to the ATP-dependent mechanism,
ATP-independent uptake of both DNP-SG and ethyl-SG was inhibited by
GSH, GSH monoethyl ester, and by low molecular weight glutathione S-conjugates (Table 2). For example, GSH and S-methyl glutathione decreased ATP-independent uptake of
[H]DNP-SG and [
H]ethyl-SG
to 53 and 33%, and 67 and 53% of control, respectively. Similarly, the
GSH analog
-Glu-AIB-Gly (ophthalmic acid) had no effect on
ATP-dependent transport but inhibited ATP-independent uptake by
30% (Table 2). Another difference between the ATP-dependent
and -independent mechanisms was observed with 100 µM DIDS
as the inhibitor. DIDS nearly completely blocked ATP-dependent
transport but only decreased ATP-independent transport to 63-73%
of control (Table 2).
Similar to the ATP-dependent mechanism,
ATP-independent uptake of DNP-SG and ethyl-SG was strongly inhibited by
BSP and relatively large or hydrophobic glutathione S-conjugates but was unaffected by glutamate, cysteine, or
dipeptides. -Glu-Cys produced a small inhibition of ethyl-SG
uptake (Table 2). In general, the various inhibitors produced
similar effects on ATP-independent ethyl-SG and DNP-SG uptake, although
the extent of inhibition was larger for ethyl-SG (Table 2).
Fig. 7illustrates the relation between the inhibitory effects
of glutathione S-conjugates and their molecular weight, using
data from Table 2. Note that the overall pattern of inhibition is
similar for both ATP-dependent and -independent transport, but there is
also a clear divergence of effects at low molecular weights. For the
ATP-dependent system, there is an abrupt increase in inhibition as the
molecular weight reaches 370 and essentially complete inhibition
at molecular weights greater than
420 (Fig. 7).
Figure 7:
Relation between the molecular weight of
transport inhibitors (glutathione S-conjugates and other
-glutamyl compounds) and ATP-dependent and -independent
glutathione S-conjugate uptake into cLPM vesicles. Data are
from Table 2. The compounds shown are, in increasing molecular
weights, 1)
-Glu-Cys, 2)
-Glu-p-nitroanilide, 3)
-Glu-Gly-Gly,
4)
-Glu-Glu, 5)
-Glu-AIB-Gly, 6)
GSH, 7) S-methyl glutathione, 8) GSH
monoethyl ester, 9) S-ethyl glutathione, 10)
glutathionesulfonic acid, 11) S-butyl glutathione,
12) S-carbamidomethyl glutathione, 13) S-carboxymethyl glutathione, 14) S-lactoyl
glutathione, 15) S-benzyl glutathione, 16) S-octyl glutathione, 17) S-(p-nitrobenzyl)-glutathione, 18) S-(p-chlorophenacyl)-glutathione, 19) DNP-SG, 20) S-ethacrynic acid glutathione, 21) GSSG, and 22)
BSP-SG.
Conjugation of electrophilic compounds with GSH functions as a key cellular defense mechanism against endogenous reactive metabolites and xenobiotics and may also be involved in inflammation, signal transmission, and modulation of cell proliferation (Boyland and Chasseaud, 1969; Chasseaud, 1979; Anderson and Meister, 1983; Ishikawa, 1992). Glutathione S-conjugates formed intracellularly must be released from the cell before further processing can occur in extracellular spaces (Hinchman and Ballatori, 1994). Current evidence indicates that the principal mechanism by which glutathione S-conjugates are transported across the cell membrane is via an ATP-dependent organic anion transporter (Kobayashi et al., 1990; Akerboom et al., 1991; Ishikawa, 1992).
The present
study demonstrates that in addition to the ATP-dependent system
glutathione S-conjugates are substrates for at least two
ATP-independent mechanisms. [H]DNP-SG uptake by
isolated cLPM vesicles was mediated by high affinity (K
8 µM) and low affinity (K
0.5
mM) ATP-independent transport components, as well as an
ATP-stimulated mechanism. Substrate specificity studies indicate that
these systems transport a wide range of glutathione S-conjugates and other anionic compounds. Although there is
considerable overlap in substrate selectivity between the ATP-dependent
and -independent systems, there are also distinct substrate
requirements (Table 2).
Glutathione S-conjugates vary
considerably in molecular size, charge, and water solubility (Boyland
and Chasseaud, 1969; Chasseaud, 1979). The present findings demonstrate
that the physicochemical properties of the S moiety are key
determinants of transport across the canalicular membrane. Relatively
low molecular weight hydrophilic conjugates are substrates for
electrogenic carriers, but not for the ATP-dependent mechanism, whereas
larger and more hydrophobic conjugates are substrates for both. Charge
also appears to be important: S-carboxymethyl glutathione
(GS-CH-COOH) decreased ATP-dependent uptake to 69% of
control, whereas S-carbamidomethyl glutathione
(GS-CH
-CO-NH
) had no effect (Table 2).
Both of these glutathione S-conjugates inhibited
ATP-independent transport of DNP-SG and ethyl-SG to a similar extent,
although the dianionic compound was a somewhat more effective
inhibitor.
DNP-SG appears to be an excellent substrate for both the
ATP-dependent and -independent systems. A comparison of the kinetic
parameters for ATP-dependent and high affinity ATP-independent DNP-SG
transport indicates that the apparent K values are
comparable (4 µM (Kobayashi et al., 1990) versus 8 µM (Fig. 5), respectively), as
are the V
values (30 versus 33
pmol
mg
10 s
),
indicating that DNP-SG should be transported at similar rates on these
competing pathways. This conclusion is supported by studies in isolated
rat hepatocytes showing that approximately 50% of DNP-SG efflux in this
experimental system is independent of ATP (Oude Elferink et
al., 1990). These investigators reported that ATP depletion to 16%
of control levels with fructose decreased DNP-SG efflux to 55% of
control. However, in contrast with the present findings, Oude Elferink et al.(1990) noted that membrane depolarization in high
K
media had no significant effect on DNP-SG efflux
from intact hepatocytes, suggesting that membrane potential is not a
driving force. However, because only a fraction of the total DNP-SG
efflux measured in intact hepatocytes is expected to be sensitive to
changes in membrane potential, the potential-sensitive component may
not have been readily detected in this experimental system.
In
contrast to DNP-SG, ethyl-SG is a poor substrate for the ATP-dependent
system and is transported almost exclusively by a high affinity
electrogenic carrier. Uptake of ethyl-SG into cLPM vesicles was only
minimally stimulated by ATP (Fig. 2), and ethyl-SG was only a
weak inhibitor of ATP-dependent DNP-SG transport (Table 2). The
absence of a low affinity ATP-independent transport component for
ethyl-SG (Fig. 6) was surprising since both DPN-SG (Fig. 5) and CHHg-SG (Dutczak and Ballatori, 1994)
exhibit high and low affinity ATP-independent systems. The reason for
this apparent discrepancy is not known but may be due to a low V
for ethyl-SG transport which would preclude
detection in the vesicle system.
Although ATP-independent transport
of glutathione S-conjugates was observed in all previous
studies of GSSG, BSP-SG, DNP-SG, and LTC transport, this
component was considered insignificant as it usually constituted less
than 50% of the total uptake or was attributed to transport by
contaminating membrane fractions. Kobayashi et al.(1990)
observed that ATP-independent DNP-SG uptake was higher in basolateral
liver membrane vesicles than in canalicular vesicles and proposed that
this electrogenic transport mechanism was localized exclusively to the
basolateral domain. Their data demonstrate that uptake of radioisotope
(from [
H]DNP-SG) is higher in basolateral
vesicles incubated in media containing either 100 mM KCl or
NaCl, as compared to sucrose, in the absence of valinomycin and ATP.
However, although the mechanism by which Na
and
K
stimulate uptake is still not clear, it cannot be
explained by membrane potential, particularly since chloride, a highly
permeant anion, was present at a concentration of 120.4 mM in
these studies. It is important to note that the experiments of
Kobayashi et al.(1990) were performed in the absence of a
-glutamyltransferase inhibitor. In our experience, there is
considerable degradation of GSH or glutathione S-conjugates by
canalicular vesicles, which are highly enriched in
-glutamyltransferase activity, unless this enzyme is inhibited.
This could contribute to the observed difference in radioisotope uptake
between canalicular and basolateral vesicles.
Early studies of GSSG
and DNP-SG transport in canalicular membrane vesicles identified low
affinity ATP-independent systems (K values of 0.4
and 1.0 mM, respectively; Akerboom et al., 1984;
Inoue et al., 1984), but these findings were subsequently
discounted when comparatively high affinity ATP-dependent systems were
described. In contrast, the present findings indicate that both
ATP-dependent and high and low affinity ATP-independent systems are
present on the canalicular membrane and most likely play a functionally
important role in biliary secretion of specific glutathione S-conjugates. The reason Akerboom et al.(1984) and
Inoue et al.(1984) failed to detect high affinity,
ATP-independent transport is explained by the relatively high substrate
concentrations used in their kinetic analyses (above 50
µM) which prevented detection of the high affinity
component (i.e.K
values of 8-10
µM; Fig. 5and Fig. 6).
Studies of BSP
and bilirubin glucuronide transport in cLPM vesicles by Nishida et
al. (1992a, 1992b) also indicate the presence of both
ATP-dependent and membrane potential-dependent organic anion transport
mechanisms. Mutant Wistar rats (TR rats) that exhibit
conjugated hyperbilirubinemia and reduced biliary excretion of certain
organic anions lack ATP-dependent BSP and bilirubin glucuronide
transport, but retain voltage-dependent canalicular transport of these
anions (Nishida et al., 1992a, 1992b). ATP-independent
bilirubin glucuronide transport has a lower V
than the ATP-dependent system (1.8 versus 17
nmol
mg
20 s
), but a
higher affinity (K
value of 26 and 71
µM), indicating that the ATP-independent system may play
an important role under physiological conditions. Because BSP,
bilirubin glucuronide, and DNP-SG are mutually competitive inhibitors
of transport, they probably share at least one transport mechanism
(Nishida et al., 1992a, 1992b). The present findings support
this hypothesis but also indicate that there may be more than one
ATP-independent organic anion transport mechanism. ATP-independent
transport of DNP-SG (Fig. 5), CH
Hg-SG (Dutczak and
Ballatori, 1994), and GSH (Ballatori and Dutczak, 1994), is mediated by
both high and low affinity components.
Although these data indicate the presence of multiple transport mechanisms, the actual number and identity of the transport proteins remains unknown. The broad and overlapping substrate specificities of the multiple transport systems, their complex kinetic behavior, and the absence of selective transport inhibitors, make it difficult to distinguish and characterize these transporters in isolated membrane vesicle systems. A complete characterization of the transporters will require their physical separation or the functional expression of the cloned cDNA.