(Received for publication, August 23, 1994; and in revised form, November 28, 1994)
From the
The transport of 2,4-dinitrophenyl-S-glutathione
(DNP-SG) into inside-out vesicles from L1210 cells was employed to
identify and characterize ATP-dependent efflux routes for DNP-SG.
Measurements of ATP-dependent uptake at varying concentrations of
[H]DNP-SG revealed the presence of two distinct
transport systems. Transport at low substrate concentrations occurred
predominantly via a high affinity system (K
= 0.63 µM), whereas a low affinity
system (K
= 450 µM)
predominated at high concentrations of substrate. The high affinity
system was characterized by a potent inhibition by the glutathione
conjugates of bromosulfophthalein (K
= 0.09 µM) and ethacrynic acid (K
= 0.44 µM),
leukotriene C
(K
=
0.20 µM), and the taurate diconjugate of bilirubin (K
= 0.10 µM). The
low affinity transport system for DNP-SG exhibited a high affinity for
bilirubin ditaurate (K
= 1.8
µM), indoprofen (K
=
3.0 µM), and biphenylacetic acid (K
= 5.9 µM). Different results were
obtained with an L1210/C7 variant which has a defect in the efflux of
methotrexate and cholate. Vesicles from the latter cells contain the
same low affinity transport activity as parental cells, but the high
affinity route is absent and has been replaced by a system with an
intermediate affinity for DNP-SG (K
= 4.5 µM). These results indicate that
L1210 cells contain two unidirectional efflux pumps for DNP-SG with
substantial differences in inhibitor sensitivity. The high affinity
system shows a binding preference for glutathione conjugates but can
also accommodate large anionic conjugates, whereas the low affinity
system has a binding preference for large organic anions. Results with
the variant cells support the hypothesis that the high affinity
transport system for DNP-SG also mediates the unidirectional efflux of
methotrexate and cholate in intact L1210 cells.
Unidirectional efflux systems are required by cells for different physiological functions that include cellular detoxification(1, 2, 3, 4, 5, 6) , antigen presentation by the major histocompatibility complex system (7) , extracellular signaling(4, 8, 9) , and the regulation of ion channels across plasma membranes(10, 11) . A representative member of this group of transporters is P-glycoprotein, which mediates the ATP-dependent expulsion of various hydrophobic drugs(1, 2, 3) . The overproduction of P-glycoprotein is often associated with multidrug resistance, and the existence of different forms of P-glycoproteins suggests that families of structurally similar transport proteins exist with complementing substrate specificities. A multidrug resistance-associated protein also contains structural elements common to P-glycoproteins(12) , and recent findings suggest that multidrug resistance-associated protein is an efflux pump for glutathione conjugates(13) .
Efflux pumps for anions exhibit substrate and inhibitor
specificities which are distinct from the P-glycoproteins overproduced
in multidrug resistance(4, 5) . Anionic efflux systems
have been described for glutathione S-conjugates(5, 14, 15, 16, 17, 18) ,
leukotrienes(4, 19, 20, 21) , cyclic
AMP(8, 9) ,
methotrexate(22, 23, 24, 25, 26, 27, 28, 29, 30) ,
and cholate(9, 25, 30) . In L1210 mouse
cells, the unidirectional efflux of methotrexate has been shown by
inhibitor studies to consist of two components which mediate 70 and 30%
of total methotrexate efflux, respectively. A variant L1210/C7 cell
line has also been isolated which lacks the primary efflux route for
methotrexate (system I) but exhibits elevated activity of the second
route (system II)(27) . System II responds generally to the
same compounds which inhibit system I, but differences exist in the
concentrations required for half-maximal efflux inhibition, and system
II does not appear to transport cholate(27) . A high
sensitivity to inhibition by BPAA ()and indoprofen is a
characteristic feature of system II.
PGA and other
nucleophiles that include CDNB and ethacrynic acid are unusually potent
inhibitors of efflux system I in L1210 cells(30) . The
effectiveness of these inhibitors was attributed to the intracellular
accumulation of the glutathione conjugates of these compounds and the
subsequent inhibition of efflux by competition with methotrexate and
cholate for binding sites on efflux proteins. The physiological
function of efflux system I was suggested to involve the expulsion of a
broad spectrum of compounds such as glutathione conjugates, large
anions, and anion conjugates(30) . The latter conclusion was
based on the ability of system I to transport anions of diverse
structure (methotrexate and cholate), its inhibition by various large
anions, and the apparent sensitivity to intracellular glutathione
conjugates.
The present study was initiated to identify and characterize unidirectional and ATP-dependent efflux systems for DNP-SG in L1210 cells and to determine whether the specificities of these systems have common features with the efflux routes for methotrexate and cholate. The L1210/C7 cell line with a defect in the efflux of methotrexate and cholate was also examined for a corresponding defect in the efflux of DNP-SG. Results using inside-out vesicles established that L1210 cells contain two ATP-dependent efflux routes for DNP-SG and that these systems differ substantially in substrate affinity and sensitivity to inhibitors. Moreover, a high affinity system for DNP-SG in parental cells was found to be defective in the efflux-deficient L1210/C7 cell line.
DNP-SG was synthesized chemically by a procedure based on the method of Vince et al.(32) . Glutathione (2.0 mmol) was dissolved in 2.0 ml of water and neutralized by the addition of 1.8 ml of 2 N NaOH (3.6 mmol). CDNB (2.4 mmol) dissolved in 15 ml of ethanol was then added dropwise to the glutathione solution, and the mixture was stirred for 16 h at 23 °C. The resulting precipitate was chilled on ice, washed with 25 ml each of ice-cold water and then ethanol, suspended in 15 ml of water, and dissolved by adjusting the pH to 7.0 with 2 N NaOH. Reprecipitation was achieved by adding 2 N HCl to pH 3.5 and chilling the sample on ice for 1 h. The final precipitate was washed with water and ethanol (as described above) and then with chloroform. DNP-SG was obtained as a dry powder in an amount corresponding to a theoretical yield of about 50% and was essentially free of CDNB as determined by HPLC (see below). Spectral analysis gave an absorbance maximum at 340 nm and a molar extinction coefficent of about 9,600.
[H]DNP-SG was
synthesized enzymatically by combining CDNB (2 µmol), glutathione
(0.25 µmol), [
H]glutathione (250
µC
), and 2 units of glutathione S-transferase
in 1.0 ml of 50 mM Tris-HCl, pH 7.4, and incubating the
mixture for 2 h at 37 °C. The incubation mixture was then diluted
with 1.0 ml of ice-cold distilled water and applied to a column of
QAE-Sephadex (1.0-ml bed volume) equilibrated with 10 mM Tris-HCl, pH 7.4. After washing successively with 2 ml of 10
mM Tris-HCl, pH 7.4, and 3 ml of water to remove the CDNB and
glutathione S-transferase, the bound
[
H]DNP-SG was eluted in 0.5-ml fractions with 0.7 N formic acid. The radioactive fractions were pooled, dried
under reduced pressure, dissolved in about 1 ml of water, redried, and
dissolved in 400 µl of water. Further purification by HPLC was
achieved by applying the sample (in 200-µl portions) to an
equilibrated 0.46
25-cm Altex C18 Ultrasphere-ODS column and
eluting in 1.0-ml fractions with a gradient of solvent A (0.1% aqueous
trifluoroacetic acid) and solvent B (0.1% trifluoroacetic acid, 10%
water, and 90% acetonitrile) according to the following program:
0-5 min, 100% A, 5-30 min 100% A to 100% B. The sharp peak
at approximately 19 min (detected at 278 nm) was collected, pooled with
the duplicate run, dried under reduced pressure, and dissolved in 1.0
ml of water. Contaminating unreacted
[
H]glutathione and oxidized
[
H]glutathione disulfide eluted in a broad peak
between 4 and 8 min under these conditions, and trace amounts of CDNB
eluted at 24 min. The concentration of [
H]DNP-SG
was determined by comparison of HPLC profiles of unknown samples and
standard solutions whose concentrations had been determined spectrally
at 340 nm (molar extinction coefficient = 9, 600). Parallel
samples subjected to scintillation counting gave specific activities in
various preparations of 425,000 ± 50,000 counts/min/nmol. The
recovery of tritium in the [
H]DNP-SG usually
exceeded 90%.
EA-SG was synthesized chemically by a procedure based
on the method of Awasthi et al.(33) . Ethacrynic acid
(0.10 mmol dissolved in 1.0 ml of ethanol) and glutathione (0.12 mmol
dissolved in 1.0 ml of 10 mM potassium phosphate, pH 7) were
combined, adjusted to pH 6.5 with 0.5 N KOH, and incubated for
24 h at 23 °C. After reducing the volume to about 0.4 ml (under
reduced pressure), the EA-SG was purified by HPLC (in 200-µl
portions) by the same procedure described above for
[H]DNP-SG. EA-SG eluted at 18 min, whereas
unreacted ethacrynic acid eluted at 23 min. After lyophilization, EA-SG
was obtained as a dry white powder in a yield of about 75%. Spectral
analysis revealed an absorbance maximum near 270 nm and an extinction
coefficient (5,700) similar to that reported by Habig et
al.(34) .
BSP-SG was synthesized enzymatically by a
procedure based on the method of Garcia-Ruiz et
al.(35) . BSP (0.10 mmol) and glutathione (0.20 mmol) were
dissolved in 1 ml of 10 mM sodium-phosphate, pH 7, and
adjusted to pH 7.5 with 0.5 N NaOH. Glutathione S-transferase (50 units) was then added, and the sample was
incubated at 23 °C for 24 h. BSP-SG was separated from unreacted
BSP and glutathione (in 200-µl aliquots) by the HPLC procedure
described above for [H]DNP-SG, except that the
gradient program was modified as follows: 0-5 min, 100% A;
5-40 min, 100% A to 30% B; 40-50 min, 30% B to 100% B.
BSP-SG eluted at 37 min, whereas BSP eluted at 44 min. BSP-SG was
obtained as a dry white powder in a yield of about 50%. BSP-SG and BSP
exhibited the same spectral properties (above 450 nm) and appeared to
have the same molar extinction coefficient (6,050) at 568 nm in 0.1 M Tris-HCl, pH 8.5.
BSP-SGSG (36) was synthesized enzymatically and purified by the same procedure as described above for BSP-SG, except that glutathione in the reaction mixture (0.50 mmol) was increased to a 5-fold molar excess over BSP (0.10 nmol), the pH was readjusted to 7.5 at 24-h intervals, and the incubation time was extended to 72 h. BSP-SGSG eluted during HPLC at 32 min and was obtained in a yield of about 15%. Concentration was determined from the extinction coefficient for BSP (at 568 nm and pH 8.5) of 6,050.
Purified vesicles incubated in
the complete assay mixture containing 1.5 µM [H]DNP-SG accumulated the labeled substrate
by a process which was linear for 10 min at 37 °C, reached a
maximum at 25 min, and then declined thereafter. Omission of the
ATP-regenerating system or ATP (1.0 mM) led to a 3- and
20-fold reduction in the transport rate, respectively, and essentially
no uptake occurred at 4 °C. A broad optimum was observed between pH
7.0 and 7.5. ATP-dependent uptake at 1.5 µM [
H]DNP-SG after 25 min at 37 °C
decreased by 1.6- and 2.4-fold when the sucrose concentration was
increased to 0.5 and 1.0 M, respectively, indicating that the
majority of uptake represented the accumulation of free substrate
within the intravesicular space and not an ATP-dependent binding of
substrate to the membrane. When the substrate concentration was
increased to 100 µM, ATP-dependent uptake of
[
H]DNP-SG remained linear for 10 min at 37
°C, and reached a maximum after 20 min.
Uptake measurements at
varying concentrations of [H]DNP-SG provided
evidence for two ATP-dependent transport components. A
double-reciprocal plot (Fig. 1) of transport at substrate
concentrations ranging from 0.10 to 500 µM was biphasic
and suggested the presence of a high and low affinity system with
substantially different kinetic properties. Curve fitting of the data
for multiple transport systems showed that the best fit was obtained
for two independent transport components with K
values for half-maximal transport of 0.63 µM (V
= 6.6 pmol/min/mg protein) (Fig. 1) and 450 µM (V
= 148 pmol/min/mg protein) (inset, Fig. 1). In a control experiment in which the transport interval
was reduced from 10 to 5 min, no significant change was observed for
the K
of the high affinity system (0.60
µM) or the low affinity system (600 µM) for
DNP-SG. Computer simulations which probed for a third transport
component with a K
between 2 and 20 µM [
H]DNP-SG produced a poor fit for the data.
Kinetic values for DNP-SG transport into L1210 cell vesicles are listed
in Table 1.
Figure 1:
Double-reciprocal plot of the substrate
dependence for [H]DNP-SG transport by inside-out
vesicles from L1210 cells. Data are shown for transport at
[
H]DNP-SG concentrations from 0.2 to 20
µM. Inset, double-reciprocal plot of transport (v) at [
H]DNP-SG concentrations (s) from 5 to 500 µM.
Transport measurements at
varying concentrations of [H]DNP-SG confirmed
that different kinetics are expressed in vesicles from the variant cell
line relative to the parent. A double-reciprocal plot of these data (Fig. 2) showed that two ATP-dependent components were present,
but the high affinity system of parental cells was absent in the
variant and had been replaced by a component with a similar V
but a reduced affinity for
[
H]DNP-SG. Computer analysis indicated that the K
for this intermediate affinity route was 4.5
µM (Table 1), a value 7-fold lower than the K
of the high affinity route in parental cells.
The low affinity system of variant cells (inset, Fig. 2) exhibited a K
and V
for DNP-SG transport which was comparable to
parental cells (Table 1), suggesting that the same low affinity
system was present in both cell lines.
Figure 2:
Double-reciprocal plot of the substrate
dependence for [H]DNP-SG transport by inside-out
vesicles from L1210/C7 cells. Data are shown for transport at
[
H]DNP-SG concentrations from 0.8 to 50
µM. Inset, double-reciprocal plot of transport (v) at [
H]DNP-SG concentrations (s) from 10 to 500
µM.
Figure 3:
Semi-log plot of the concentration
dependence for inhibition of [H]DNP-SG transport
in vesicles from L1210 cells by various compounds. Measurements were
performed at 1.5 µM [
H]DNP-SG. MTX, methotrexate.
Large anionic
compounds which are not glutathione conjugates were also examined for
the ability to inhibit the high affinity transport system for DNP-SG (Table 2). The most potent of these compounds was the taurate
diconjugate of bilirubin, whose K of 0.10
µM was comparable to the most effective glutathione
conjugates. QUIN-2 (a tetraanionic quinoline derivative), bilirubin,
BSP, and indomethacin inhibited with an intermediate affinity, whereas
other aromatic anions (e.g. BPAA and indoprofen) were poor
inhibitors of this system. An unusual response was observed for
cholate, which produced an increase in transport to 125% of the control
at 50 µM, a subsequent decline to control levels at 150
µM, and inhibition by 50% at 350 µM (data not
shown). Methotrexate also inhibited the high affinity system for DNP-SG (Fig. 3) but had a relatively low affinity (K
= 300 µM).
BPAA (Fig. 4A),
flurbiprofen, and indoprofen inhibited [H]DNP-SG
transport (at 1.5 µM) in parental vesicles, but the extent
of inhibition reached only 10% at concentrations below 20 µM and then remained relatively constant thereafter. This latter
result suggested that these compounds had inhibited the portion of
total uptake representing the low affinity system, but had little or no
effect on the high affinity system.
Figure 4:
[H]DNP-SG transport
in vesicles from L1210 cells at varying concentrations of BPAA in the
absence (A) and presence of EA-SG (B). A,
response of transport at 1.5 µM [
H]DNP-SG to increasing concentrations of
BPAA. B, response of transport at 5.0 µM [
H]DNP-SG to increasing concentrations of
BPAA in the presence of a fixed concentration (40 or 100
µM) of EA-SG. Transport in the controls without inhibitor
were 6.1 and 8.6 pmol/min/mg protein at 1.5 and 5.0 µM [
H]DNP-SG,
respectively.
Isolation of
the low affinity system was a more challenging problem since this route
contributed a majority of total transport only at relatively high
substrate concentrations. To improve the relative contribution from
this route, the substrate concentration was raised to 5.0
µM, and EA-SG was added (at 40 µM) to inhibit
the high affinity system. The resulting ATP-dependent transport
activity for [H]DNP-SG was then evaluated for a
BPAA-sensitive transport component (Fig. 4B). Under
these conditions, maximal inhibition by BPAA reached 50%, and the
concentration for half-maximal inhibition was 6 µM (K
= 5.9 µM). At 100
µM EA-SG, the BPAA-sensitive portion of
[
H]DNP-SG transport increased to 75%, and the
IC
for BPAA remained at 6 µM (Fig. 4B). Using the latter conditions, K
values for the low affinity system were also
obtained for indoprofen (K
= 3.0
µM), indomethacin (K
= 10
µM), and bilirubin dituarate (K
= 1.8 µM) (Table 2). Hence, EA-SG could
be shown to be a potent and relatively specific inhibitor of the high
affinity transport system, but conditions were not achieved for a
complete separation of the two transport activities. Even at 100
µM EA-SG, interference from the high affinity system was
approximately 25%.
Figure 5:
[H]DNP-SG transport
at increasing concentrations of EA-SG (A) or BPAA (B)
in vesicles from L1210/C7 cells. [
H]DNP-SG
concentrations, 5.0 µM; transport in the control without
inhibitor, 4.0 pmol/min/mg protein.
DNP-SG transport in L1210/C7 vesicles in the presence of combinations of EA-SG and BPAA is shown in Fig. 6. Transport at a fixed concentration of EA-SG (40 µM) and varying levels of BPAA is shown in Fig. 6A. The resulting inhibition by BPAA appeared monophasic and extrapolated to greater than 90% at high levels of BPAA. Similarly, transport at a fixed concentration of BPAA (100 µM) and varying levels of EA-SG produced a similar monophasic profile and a maximum inhibition that also exceeded 90% (Fig. 6B). Hence, EA-SG and BPAA can be employed for a nearly quantitative separation of the intermediate and low affinity transport systems in L1210/C7 vesicles.
Figure 6:
Inhibition of
[H]DNP-SG transport by combinations of EA-SG and
BPAA in vesicles from L1210/C7 cells. Transport at 5.0 µM [
H]DNP-SG was measured in the presence of 40
µM EA-SG (+EA-SG) (A) and varying
concentrations of BPAA or 100 µM BPAA (+BPAA) (B) and varying concentrations of
EA-SG.
The
inhibitor specificity of the transport systems for DNP-SG in L1210/C7
vesicles was determined by measuring inhibitor responses in the
presence of 100 µM BPAA or 40 µM EA-SG.
[H]DNP-SG transport in the presence of a constant
level of EA-SG or BPAA and increasing concentrations of three selected
inhibitors is shown in Fig. 7. Flurbiprofen (Fig. 7A) mediated a potent inhibition of DNP-SG
transport in the presence of EA-SG, but not BPAA, indicating that only
the low affinity system was sensitive to this inhibitor. Conversely,
BSP-SG (Fig. 7B) was a potent inhibitor of
[
H]DNP-SG transport in the presence of BPAA, but
was much less effective with added EA-SG, indicating that the
intermediate affinity system was more sensitive to this inhibitor.
Bilirubin ditaurate (Fig. 7C) inhibited transport in
the presence of either BPAA or EA-SG.
Figure 7:
Response of the intermediate and low
affinity transport systems of L1210/C7 cells to (A)
flurbiprofen (FB), (B) BSP-SG, and (C)
bilirubin ditaurate (BDT). Transport by the intermediate
affinity system was determined at 5.0 µM [H]DNP-SG in the presence of 100 µM BPAA (+BPAA), whereas the low affinity system was
determined in assay mixtures containing 40 µM EA-SG (+EA-SG).
Various compounds and their K values for inhibition of the intermediate and
low affinity transport systems for DNP-SG in L1210/C7 vesicles are
shown in Table 3. These two transport systems responded to
different types of compounds, although an overlap in specificity was
evident with some inhibitors. The intermediate affinity system of
L1210/C7 cells responded generally to the same compounds which
inhibited the high affinity system of parental cells. The most
effective compounds were glutathione conjugates, bilirubin ditaurate,
QUIN-2, bilirubin, and BSP, whereas GS-SG and various monovalent
aromatic anions were poor inhibitors of this system. K
values for unlabeled DNP-SG coincided with K
values for [
H]DNP-SG transport. Inhibition
was also obtained with methotrexate (K
=
220 µM), whereas cholate produced a stimulation similar to
that observed for the high affinity system. The extent of this
stimulation was somewhat higher in L1210/C7 vesicles (50% at 50
µM cholate), although the general pattern was the same;
stimulation at low levels of cholate, a maximum at 50 µM,
and inhibition at higher concentrations. A double-reciprocal plot of
transport at varying concentrations of [
H]DNP-SG
showed that 50 µM cholate increased the V
for the intermediate affinity system by 50% but had little effect
on the K
(4.0 µM) for DNP-SG (data
not shown).
The low affinity system for DNP-SG in vesicles from
L1210 cells exhibited a high affinity for several large anions (Table 3). The most effective compound was bilirubin ditaurate (K = 1.5 µM), although strong
inhibition (K
= 3.0-6.0
µM) was also observed with monovalent anions (indoprofen,
flurbiprofen, and BPAA) which were not inhibitors of the high or
intermediate affinity systems (cf. Table 2and Table 3). Bilirubin (K
= 40
µM) and methotrexate (K
= 150
µM) were moderate inhibitors of this transport system. The
most pronounced inhibition by a glutathione conjugate was observed with
BSP-SG, but the K
for BSP-SG (60 µM)
was indistinguishable from the K
for BSP alone (50
µM). Inhibition by unlabeled DNP-SG (at concentrations up
to 1.0 mM) produced a K
(450
µM) which was consistent with the observed K
(500 µM) for
[
H]DNP-SG. Analogs of BPAA with decreasing
molecular size (naphthalene acetic acid and phenyl acetic acid) were
progressively weaker inhibitors of the low affinity system.
L1210 mouse cells contain two ATP-dependent transport systems
which mediate the efflux of DNP-SG. These systems were detected and
characterized using inside out vesicles which had been purified from
isolated plasma membranes. The two systems can be readily distinguished
by their substantially different K values for
half-maximal transport of [
H]DNP-SG (0.63 and 450
µM, respectively) and by differences in relative
contribution at different substrate concentrations. Transport at DNP-SG
concentrations below 1 µM proceeds almost exclusively by
the high affinity system, whereas these two systems contribute
approximately equally to total transport when the concentration of
DNP-SG is raised to 30 µM. Numerical values for K
that had been determined for each route by
influx analysis (Table 1) were confirmed by inhibitor analysis
with unlabeled DNP-SG ( Table 2and Table 3). The present
finding of multiple transport routes for DNP-SG in L1210 cells is
consistent with prior studies in which human erythrocytes were also
shown to contain two energy-dependent efflux routes with substantially
different K
values for DNP-SG. K
values of 1.4 and 700 µM were detected in intact
cells(42) , whereas slightly higher K
values of 3.9 and 1,600 µM were observed in
inside-out vesicles(43) . High affinity, ATP-dependent efflux
systems for DNP-SG with a K
of 4 and 20
µM, respectively, have also been demonstrated in rat liver
canalicular membrane vesicles (15) and in vesicles from rat
heart sarcolemma(14) .
The inhibitor specificity of the high
affinity transport system in L1210 vesicles was determined with minimal
interference from the low affinity route by employing a low
concentration of [H]DNP-SG. The most effective
inhibitor of this system was BSP-SG, whose affinity (K
= 0.09 µM) was 7-fold higher than DNP-SG (K
= 0.63 µM). Other large
anionic conjugates (BSP-SGSG, LTC
, EA-SG, and bilirubin
ditaurate) were also bound with a higher affinity than DNP-SG, whereas
a lower affinity was observed for S-hexyl glutathione and
GS-SG. Glutathione conjugates with aromatic (BSP-SG) or aliphatic
(LTC
) substituents were accommodated with a comparably high
affinity, and conjugation with glutathione was not required for binding
since a high affinity (K
= 0.10
µM) was also observed for the taurate diconjugate of
bilirubin. The same high affinity observed in the present study for
LTC
(K
= 0.20 µM)
had also been observed for LTC
transport via a glutathione
conjugate pump in membrane vesicles from rat heart sarcolemma (K
= 0.15 µM) and rat liver (K
= 0.25 µM)(20) .
Our competition studies support the model of Awasthi (5, 44) in which the substrate binding site on the
high affinity glutathione conjugate pump is suggested to have a
relatively broad specificity. Using the designations of
Ishikawa(4, 45) , the G-domain for binding of the
glutathione portion of conjugates appears to accept both the
zwitterionic glutathione structure as well as the anionic composition
of taurate diconjugates, and the C-domain for the adduct portion of
conjugates accommodates uncharged compounds with an aromatic
dinitrophenyl or aliphatic S-hexyl group, as well as anionic
compounds with large aliphatic (LTC
) or aromatic (BSP)
constituents. A high affinity for the glutathione diconjugate of BSP is
a further indication of substantial flexibility at the substrate
binding site.
Transport systems involved in cellular detoxification
would be expected to possess a general ability to translocate various
competitive inhibitors which exhibit structural similarities to known
substrates (e.g. DNP-SG), and in some cases, this possibility
has been confirmed by direct measurements. Transport of LTC and DNP-SG has been determined in rat liver plasma
membranes(20) , and each compound was deduced from kinetic
studies to be a substrate of the same transport system. The high
affinity system for DNP-SG in L1210 vesicles is inhibited by various
structurally similar glutathione conjugates and by other compounds (e.g. bilirubin ditaurate) which may also be transported by
this system, although this has not been verified by direct
measurements. Compounds with a relatively low affinity for this system
may also be transport substrates. Regardless of affinity, efficient
transport of inhibitors could occur as long as the V
increases proportionally with decreasing affinity.
Prior
kinetic studies have suggested that methotrexate may exit L1210 cells
via systems which also function in the extrusion of glutathione
conjugates(30) . This latter conclusion was based, in part, on
the potent inhibition of methotrexate efflux by compounds, such as
prostaglandin A, ethacrynic acid, and CDNB, which can be
readily converted to intracellular glutathione conjugates. The present
study shows that methotrexate is an inhibitor of the high affinity
carrier for DNP-SG and hence supports the notion that methotrexate may
utilize the latter system to exit L1210 cells. The relatively low
affinity of the conjugate pump for methotrexate (K
= 300 µM) does not detract from this model
since a low affinity for methotrexate had been suggested from prior
studies. Methotrexate efflux showed no evidence of saturation in intact
L1210 cells which had been loaded with methotrexate to internal
concentrations up to 30 µM (120 pmol/mg protein). (
)The possibility that methotrexate efflux occurs via a
glutathione conjugate pump is also supported by measurements in
inverted human erythrocyte ghosts(46) . Methotrexate transport
in this system was inhibited by several compounds which also inhibit
glutathione conjugate transport. Moreover, the reported K
for methotrexate transport (470 µM)
was similar to the K
observed in the present study
(300 µM) for inhibition of the high affinity system by
methotrexate.
The proposal that methotrexate efflux by system I in intact cells occurs via the high affinity efflux system for DNP-SG is further supported by transport studies using vesicles from L1210/C7 cells. The loss in ability to efflux methotrexate by L1210/C7 cells (27) is accompanied by a corresponding loss in the ability of L1210/C7 vesicles to transport DNP-SG (cf.Fig. 1and Fig. 2), and the defect was traced to the high affinity transport system for DNP-SG.
Vesicles from L1210/C7 cells contain an
intermediate affinity transport route for DNP-SG which could not be
detected in the parental cells. This additional system may be a mutated
form of the high affinity route of parental cells since these systems
exhibit a comparable V for DNP-SG transport (Table 1), and both have a similar binding preference for
glutathione conjugates (cf. Table 2and Table 3).
Alternatively, the intermediate affinity system may represent a
separate route which had been induced or stimulated from low levels in
the variant, or had been obscured in the parent by interference from
the high affinity system.
The low affinity transport system for
DNP-SG was isolated by performing transport measurements in the
presence of a specific inhibitor of the higher affinity route. EA-SG
(at concentrations of 40-100 µM) substantially
reduced transport via the high affinity system in parental vesicles (Fig. 4) and essentially eliminated interference from the
intermediate affinity system in L1210/C7 vesicles ( Fig. 5and Fig. 6). Parental and variant cells appear to express the same
low affinity transport system for DNP-SG since no differences were
observed in the K or V
for
DNP-SG transport (Table 1), and these two routes exhibited the
same response to inhibition by BPAA, indoprofen, bilirubin ditaurate,
and indomethacin (cf.Table 2and Table 3).
The
inhibitor specificity of the low affinity transport system for DNP-SG
suggests that zwitterionic glutathione conjugates are not the primary
efflux substrates for this system. Affinity measurements (Table 3) indicate that this system is an organic anion pump
since it has a relatively high affinity for large monovalent anions
(BPAA or indoprofen), anionic conjugates (bilirubin ditaurate), and
large divalent anions (bilirubin and BSP). A preference for conjugated
divalent anions is indicated since this system exhibits a 40-fold
higher affinity for bilirubin ditaurate relative to bilirubin. Since
BSP and BSP-SG interact with about the same affinity, the glutathione
constituent of BSP-SG does not appear to enhance or hinder the binding
of BSP. The minimum size requirement for anion binding is about 200
Daltons, as determined from the increase in affinity with size in a
series of structurally related monovalent anions: phenylacetic acid (K >200 µM); naphthalene acetic
acid (K
= 70 µM); and
biphenylacetic acid (K
= 5.5
µM). Candidates for physiological substrates of this anion
efflux system include various mono- and diglucuronide conjugates and
anionic mercapturates derived from the metabolism of zwitterionic
glutathione conjugates.
The low affinity efflux system for DNP-SG
may also be the same system which mediates the efflux of methotrexate
by system II in intact L1210 cells(26, 27) .
Methotrexate structurally fits the general specificity of this system
for large organic anions and was shown to bind with a moderate affinity (K = 150 µM). The primary
evidence linking these two transport systems is the close correlation
in the ability of BPAA, indoprofen, and flurbiprofen to inhibit both
the low affinity transport system for DNP-SG in vesicles (see Table 2and Table 3) and efflux system II for methotrexate
in intact cells(26, 27) .
Electrophiles are usually detoxified in mammalian cells via a glutathione S-transferase dependent conjugation with glutathione and subsequent elimination of the hydrophilic products via efflux pumps. Endogenous compounds may also require conjugation without the involvement of glutathione and excretion of the anionic products. Bilirubin ditaurate is representative of the latter group of compounds. Since xenobiotics, electrophiles, and endogenous compounds programmed for catabolism may exhibit a variety of possible structures, detoxifying systems must be able to accommodate a range of possible anions and anion conjugates to protect cell integrity. Glutathione S-transferases contribute to this diversity by comprising a family of enzymes with differing specificities(47) . Similarly, the extrusion of unwanted anions and zwitterions of various sizes and structures may be achieved most efficiently by a combination of efflux pumps of varying specificities. The notion of multiple efflux routes for glutathione conjugates is supported in the present study with L1210 cells and previously in human erythrocytes. The latter contain high and low affinity efflux systems for DNP-SG(42, 43) , and a separate activity for the efflux of oxidized glutathione(14) . Multiple anion efflux systems have also been demonstrated in intact L1210 cells by the presence of two unidirectional efflux routes for methotrexate(26, 27) . An inhibitor analysis of the high and low affinity transport systems for DNP-SG in L1210 cells shows that these two effux systems, in combination, accommodate and presumably extrude a broad range of structurally diverse compounds with a molecular requirement which includes only a moderate-to-large size and a net negative charge.