Novel properties of hepatic canalicular reduced glutathione
transport revealed by radiation inactivation
Aravind V.
Mittur1,2,
Neil
Kaplowitz1,
Ellis S.
Kempner3, and
Murad
Ookhtens1
1 Liver Disease Research
Center, Department of Medicine, and
2 Department of Biomedical
Engineering, University of Southern California, Los Angeles, California
90033; and 3 Laboratory of
Physical Biology, National Institute of Arthritis and
Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892
 |
ABSTRACT |
Transport of GSH at the canalicular pole of
hepatocytes occurs by a facilitative carrier and can account for
~50% of total hepatocyte GSH efflux. A low-affinity unit with
sigmoidal kinetics accounts for 90% of canalicular transport at
physiological GSH concentrations. A low-capacity transporter with high
affinity for GSH has also been reported. It is not known whether the
same or different proteins mediate low- and high-affinity GSH
transport, although they do differ in inhibitor specificity. The bile
of rats with a mutation in the canalicular multispecific organic anion
transporter (cMOAT or MRP-2, a 170-kDa protein) is deficient in GSH,
implying that cMOAT may transport GSH. However, transport of GSH in
canalicular membrane vesicles (CMV) from these mutant rats remains
intact. We examined the functional size of the two kinetic components
of GSH transport by radiation inactivation of GSH uptake in rat hepatic
CMV. High-affinity transport of GSH was inactivated as a single
exponential function of radiation dose, yielding a functional size of
~70 kDa. In contrast, low-affinity canalicular GSH transport
exhibited a complex biexponential response to irradiation,
characterized by an initial increase followed by a decrease in GSH
transport. Inactivation analysis yielded a ~76-kDa size for the
low-affinity transporter. The complex inactivation indicated that the
low-affinity transporter is associated with a larger protein of ~141
kDa, which masked ~80% of the potential transport activity in CMV.
Additional studies, using inactivation of leukotriene
C4 transport, yielded a functional
size of ~302 kDa for cMOAT, indicating that it functions as a
dimer.
biliary transport; target analysis; canalicular multispecific
organic anion transporter
 |
INTRODUCTION |
REDUCED GLUTATHIONE (GSH), the most abundant endogenous
thiol in virtually all cells, is maintained at millimolar (approximate micromoles per gram tissue) intracellular concentrations (24). It plays
a major role in detoxification of xenobiotics and regulation of the
thiol-disulfide status of cellular proteins. In addition it serves as a
storage and transfer vehicle for cysteine. Hepatic release of GSH into
the sinusoidal plasma and the canalicular bile undergoes a maturational
change characterized by the biliary fraction rising from ~5 to
~50% of the total efflux (25).
Although sinusoidal efflux of GSH is well understood (16, 26, 28), the
significance of canalicular efflux and its high concentration in bile
(mM) is unclear. Biliary GSH has been hypothesized to be a secretagogue
for bile acid independent bile flow (4), although the importance and
purpose of bile acid independent bile flow are unknown. Cysteine in the
biliary tract, liberated by the hydrolysis of GSH, may be reabsorbed
from the intestine and utilized for the resynthesis of GSH. The
intestine may also reabsorb intact GSH through a
Na+-dependent transport mechanism
(11). Thus the biliary efflux of GSH may have a role in
hepato-bilio-enteric circuit similar to that of sinusoidal efflux in
the hepatosystemic circulation (26, 28).
Studies of the kinetics of biliary GSH efflux in isolated perfused
livers are limited because hepatic GSH concentrations cannot be raised
more than two- to threefold above in vivo levels by typical treatments
(17, 25). However, the canalicular membrane-enriched vesicle (CMV)
preparation is a suitable model that has been used extensively to
characterize the canalicular GSH transport system (2, 7, 9, 10, 14,
27). Canalicular transport of GSH is mediated by two kinetic
components: one of high affinity with Km of
~100-200 µM and one of low affinity with
Km ~14-17 mM for GSH. The high-affinity component is governed by
Michaelis-Menten kinetics, whereas the low-affinity component is
characterized by sigmoidal kinetics with a Hill coefficient of
n ~2 (27, 21). The latter implies
allosterism and positive cooperativity between multiple binding sites
on a monomer or interactions between subunits in an oligomeric
transport system.
In two mutant rat strains, TR
and EHBR,
hyperbilirubinemia is characterized by the hepatic retention of certain
organic anions and their conjugates due to deficient secretion. This
defect has been attributed to the absence of canalicular multispecific
organic anion transporter (cMOAT or MRP-2, a 170-kDa protein), from the plasma membrane, due to a point mutation in this protein (15, 29).
Unlike normal rats, these mutant rats do not have GSH in their bile,
implying that cMOAT may be the principal transporter for biliary GSH
(15, 29). However, the transport of GSH in CMV from EHBR rats remains
intact, suggesting the existence of a distinct GSH transporter (10). An
initial attempt to clone this transporter was reported (35), but
further observations have shown the clone to be artifactual and
indicative of a bacterial protein.
The in situ functional size of the canalicular GSH transport system is
unknown. Thus it is not known whether the functional size of the
canalicular GSH transport system is similar to the molecular size of
cMOAT (15, 29) or whether the GSH transport system associates with
other proteins. It is also not known whether the two kinetic components
of canalicular GSH transport (with distinct affinities and kinetic
features) are mediated by common or different proteins.
We used the radiation inactivation technique and target size analysis
(13) in the CMV model to estimate the functional size(s) of the
protein(s) that mediates canalicular GSH transport. Additional studies,
using a specific substrate of cMOAT, i.e., leukotriene (LT)
C4, were done to compare the in
situ characteristics of GSH transporter(s) and cMOAT and their
estimated sizes in our experimental model. Radiation inactivation is a
powerful tool that has been applied to determine the functional
molecular sizes of enzymes, receptors, and transport systems (13, 19).
The distinct characteristics of the kinetics of canalicular GSH
transport, namely the negligible cross-contributions of high- and
low-affinity components to total transport at the extremes of GSH
concentrations, presented us with a unique opportunity to functionally
differentiate and study the two kinetic components.
In this study, we have attempted to answer the following two questions.
1) What are the in situ functional
sizes of the proteins responsible for the high- and low-affinity
transport of GSH across the canalicular membrane?
2) How do the functional sizes of
the GSH transport systems compare with the putative size of cMOAT?
 |
MATERIALS AND METHODS |
Chemicals and assays.
All chemicals were purchased from Sigma (St. Louis, MO) or other
reputable commercial sources.
[35S]GSH (>100
Ci/mmol),
[3H]taurocholic acid
(>2 Ci/mmol),
L-[3H]glutamate
(>40 Ci/mmol)
[3H]LTC4
(>110 Ci/mmol), and Aquasol scintillation cocktail were procured from
NEN (Boston, MA). Membrane filters (HAWP 02500) were obtained from
Millipore (Bedford, MA). The purity and molecular form of each lot of
[35S]GSH were
confirmed by HPLC (8), using a Shimadzu binary system (LC-10AT pumps
and SPD-10A controller), equipped with a UV-VIS detector (SPD-10A) and
an in-line
-RAM radiodetector (IN-US Systems). Membrane filters were
dissolved in 10 ml Aquasol, and the radioactivity was counted in a
Beckman LSC6000 liquid scintillation spectrometer. Spectrophotometric
assays were performed on a Beckman DU 7400 diode array
spectrophotometer. An HPLC method was used to assay 5'-nucleotidase as described (1).
Animals.
Mature (90-135 days old) male Sprague-Dawley rats weighing
380-450 g were purchased from Harlan Laboratories (San Diego, CA) and housed in a constant temperature and humidity environment with
alternating 12:12-h light-dark cycles. The rats had free access to
water and were fed Purina Rodent Chow ad libitum.
Isolation, characterization, and handling of membrane vesicles.
CMV were prepared by the method of Meier et al. (23) with minor
modifications. Purity and enrichment of membrane fractions were
determined by marker enzymes as described previously (10). Vesicles
were suspended in cryoprotective buffer (14% glycerol, 1.4%
D-sorbitol, 150 mM KCl, 0.2 mM
CaCl2, 0.2 mM
MgCl2 in 10 mM HEPES-Tris buffer,
pH 7.5). Aliquots of 200 µl were separated in glass ampoules (2 ml;
12011L-2, Kimble), rapidly frozen with liquid nitrogen, sealed with an
O2-gas torch, and maintained at
80°C, except during irradiation at
135°C.
Radiation dosimetry and sample handling were monitored and validated by
inactivation of the apical enzyme, 5'-nucleotidase, as an
internal control in every irradiated preparation of CMV (18).
Transport measurements.
Transport of radiolabeled substrates was determined with a rapid
filtration technique (22). Frozen canalicular membranes were quickly
thawed at 37°C and vesiculated by 15 passages through a 25-gauge
needle. The suspension was diluted in the cryoprotective buffer
(2-3 mg protein/ml) and mixed by five additional passes through a
25-gauge needle.
Aliquots of CMV (20 µl, 30-50 µg protein) were preincubated (3 min, 25 or 37°C) and uptake of radiolabeled substrates was initiated by addition of 80 µl of incubation buffer containing the
radiolabeled substrate and an appropriate driving force, when required
(e.g., ATP for LTC4 and
taurocholate). Uptake was terminated by the addition of 2 ml ice-cold
stop buffer. Stop buffers were identical in composition to incubation
buffers, without the substrate or driving force.
Transport of GSH (7, 10) and LTC4
was determined under voltage-clamped conditions at 25 and 37°C,
respectively, similar to the uptake of
L-glutamate and taurocholate.
Tracer [35S]GSH was
supplemented with unlabeled GSH to arrive at final concentrations of
0.02 µM (trace) to 50 mM GSH. Transport of
LTC4 was studied at 1 and 5 µM.
Initial rates of uptake were measured at 10 (GSH) and 15 s
(LTC4) under
zero-trans conditions by terminating
the incubations with addition of 2 ml ice-cold stop solution. The selected sampling times were in the linear range of the time-dependent uptake of each compound. "Nonspecific" binding was estimated by the radioactivity bound to CMV at 4°C and 0 time, determined by adding a chilled aliquot of CMV to
2 ml ice-cold stop solution. The net initial rate of uptake
(nmol · mg
1 · 10 s
1) was calculated by
subtracting the nonspecific binding from the 10- and 15-s uptakes.
Uptake of GSH was determined under voltage-clamped conditions in CMV
resuspended at a final concentration of 75 mM K+ and treated with valinomycin
dissolved in ethanol (10-12 µg/mg protein) for 1 h at 25°C.
Uptake of
[3H]taurocholate (~1
µM) was studied at 37°C under voltage-clamped conditions in an
incubation buffer that contained a final concentration of 75 mM
K+ and 5 mM ATP (5). The stop
buffer also contained 75 mM K+.
The ATP-dependent fraction in LTC4
transport was obtained from the difference of uptake in the absence and
presence of 4 mM ATP in an ATP regenerating system.
The presence of significant ATP-dependent uptake of taurocholate and
sodium-dependent uptake of glutamate was used as a benchmark for the
functional integrity of CMV suspended in cryoprotective buffer. CMV
preparations that failed to demonstrate adequate activity of these two
transport systems were not used.
Radiation inactivation and target size analysis.
Aliquots of CMV preparations were irradiated at
135°C with
10 MeV electrons from a linear accelerator (Armed Forces Radiobiology Research Institute, Bethesda, MD) as described (13). Analysis of the
surviving activity in radiation inactivation studies has been described
elsewhere (13, 20). The exponential decay in the activity with
radiation dose (in rad), AD = A0
e-µD, is used
for target analysis to establish the target or functional size of
proteins using the following relationship
where µ is the rate constant of the exponential representing the
inactivation dose-response curve and M is the functional size of the
protein in kilodaltons.
Postirradiation residual transport activity
(V) was normalized to the transport
activity in the unirradiated control
(V0) and
expressed as
%V/V0.
The rate constant of the inactivation curve [slope of
ln(V/V0)
against radiation dose, D] was estimated by fitting the data from
both individual experiments separately and by fitting the mean of
residual activities, statistically weighted by their respective
variances in each group. In cases where a radiation-insensitive
component (Z) of activity was observed, the data were analyzed as
described (27) and fitted with
In
cases where a complex biphasic radiation response was observed, i.e., a
rise and fall in surviving transport activities (see Fig. 4), the data
were fitted with the difference of two exponentials (20)
where
V is transport activity measured in
samples exposed to radiation dose D,
A0 is total potential transport
activity in unirradiated samples, and µ is rate constant of the
terminal exponential decay of the inactivation curve, proportional to
the functional mass of the transporter, as indicated above; Z is the
same as above; B0 equals
A0
100;
is the rate
constant of the exponential defined by the difference between the
terminal exponential and the data (i.e.,
A0
e-µD minus the
observed transport at dose D).
Kinetic analysis and fitting of data.
Kinetics of transport were analyzed using the sum of a Michaelis-Menten
and a sigmoidal Hill (n ~2)
component
where
V is the total net transport rate of
GSH by the entire transport system.
The
fits to the kinetic as well as the irradiation data were obtained using
the SAAM II program (version 1.0.3 for Macintosh) (32) and a nonlinear,
least-squares error criteria. Data from individual experiments were
fitted by weighting the points equally, whereas SEs were used to weight
the mean values of the pooled data from each group.
 |
RESULTS |
Enrichment, purity, and functional integrity of membrane vesicles.
The CMV fractions were enriched 23-fold in
Mg2+-ATPase and 55-fold in
alkaline phosphatase but deenriched or not enriched in intracellular
organelle markers (Table 1). Each CMV
preparation was essentially free of cross-contamination by sinusoidal
membranes, as indicated by the negligible activity of the basolateral
marker Na+-K+-ATPase.
All seven vesicle preparations showed the characteristic increase in
the uptake of
[3H]taurocholate
driven by ATP (22), confirming the functional integrity of CMV (data
not shown). The Na+-dependent
transport of
L-[3H]glutamate
(2 µM and 50 mM) in the presence of a
K+ gradient was used as a marker
of passive permeability in three CMV preparations as an additional
check for integrity of vesicles. Transport of glutamate (2 µM) in the
absence of Na+ (3) was negligible
and did not change with irradiation dose, further confirming the
functional integrity of the CMV preparations (data not shown).
Inactivation of 5'-nucleotidase.
We estimated the functional size of 5'-nucleotidase as an
internal control to monitor the dosimetry and consistency of
irradiations in individual vesicle preparations. In seven independent
preparations, the inactivation of this enzyme yielded a target size of
83 ± 2 (SE) kDa, in excellent agreement with the known structure
and previous radiation inactivation analyses (18). The mean of residual activities pooled from seven individual preparations is shown in Fig.
1 along with the fitted single exponential
that defines the decay.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Inactivation of hepatic 5'-nucleotidase. Residual activity in 7 independent preparations were pooled, and mean values are shown ( ).
Solid curve represents fit of monoexponential function to data weighted
by SEs, represented by the bars (see target size in text).
|
|
Kinetics of GSH transport in unirradiated vesicles.
We verified that the kinetics of GSH uptake in the presence of the
cryoprotective buffer was similar to that observed in standard suspension buffers reported in past studies (2, 7, 9, 10, 14, 27). The
kinetics of GSH uptake in four to seven independent CMV preparations
suspended in the cryoprotective buffer is presented in Fig.
2. [Voltage-clamped and
nonvoltage-clamped determinations of kinetics were indistinguishable
(not shown).] The solid lines in Fig. 2 represent the fits to the
data using the model presented in MATERIALS AND
METHODS and are representative of high- (i.e., 150 µM) and low-affinity (i.e., 16 mM) average Km values
reported in past studies (2, 7, 9, 10, 14, 27). As can be seen, the
kinetics measured in the cryoprotective buffer was not different from
that in noncryoprotective buffers. The broken lines in Fig. 2 show the
contributions of the high- and low-affinity components to overall
transport.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Kinetics of GSH transport in canalicular membrane vesicles. Data
represent means ± SD of uptake in 4-7 independent preparations
suspended in cryoprotective buffer. Initial rates of
[35S]GSH uptake (10 s)
were measured under voltage-clamped conditions at concentrations
ranging from 0.02 µM (trace) to 50 mM GSH. Solid lines indicate fit
to overall transport obtained by the sum of 2 components,
Michaelis-Menten (high affinity) and Hill (low affinity). B
shows high-affinity region on expanded scale. Broken and dotted lines
indicate contributions by high- and low-affinity components,
respectively. Km values used
were representative of those from past studies conducted in
noncryoprotective buffers (see text).
A:
Km = 16.0 mM.
B:
Km = 0.15 mM.
There were no significant differences between kinetics determined in
either medium. Kinetics indicate that >99% of transport at trace and
0.02 mM GSH is by high-affinity component, whereas >95% of transport
at 25 and 50 mM GSH is by low-affinity component.
|
|
Radiation inactivation of GSH transport.
The kinetics of GSH transport in CMV (Fig. 2) indicates that >99% of
total transport at trace (0.02 µM) and 0.02 mM GSH is mediated by the
high-affinity component. Therefore, the functional size of this
component was determined by radiation inactivation of uptake at these
two concentrations of GSH. Radiation exposure caused a decay in
residual transport down to ~10% of that in the unirradiated samples,
but further exposure (>65 Mrad) had little effect on uptake (Fig.
3, A and
B).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Inactivation of high-affinity transport of GSH. Residual transports at
trace GSH (A, ) in 4 independent
preparations and at 0.02 mM GSH (B,
) in 7 independent preparations are shown. , Inhibited values of
residual GSH transport in presence of 5 mM disulfobromophthalein (mean
of duplicate determinations from up to 2 preparations). Solid lines
indicate fits obtained using monoexponential function plus constant
value of 7% (see text) weighted by SEs, represented by the bars (see
target sizes in text).
|
|
The radiation-insensitive residual transport is due to nonspecific
factors, such as binding and/or passive diffusion. Experimental verification of this point was made with disulfobromophthalein (diBSP),
an organic anion that inhibits canalicular GSH transport (Ref. 2 and
Mittur and Ookhtens, unpublished observations). Addition
of 5 mM diBSP reduced GSH uptake in both unirradiated and irradiated
samples from the levels observed in the uninhibited controls down to a
mean value of ~7% at both trace and 0.02 mM GSH (Fig. 3,
A and
B,
). Thus nonspecific activity
accounts for most of the residual transport at the highest radiation
exposures. Therefore, the pooled data were fitted with the sum of an
exponential plus a constant, representing the noninhibitable fraction
equal to ~7% of the unirradiated transport. The functional sizes for the high-affinity GSH transport were estimated to be 73 ± 5 (SE) kDa at trace GSH and 69 ± 5 (SE) kDa at 0.02 mM GSH (fits to pooled data, weighted by SEs, shown with solid curves in Fig. 3,
A and B). No differences in the estimated
mean target sizes were found when the data from individual experiments
were fitted separately (Table 2), compared
with the estimates obtained by fitting the pooled data.
The functional size of the low-affinity component of GSH transport was
determined at 25 and 50 mM GSH, at which it accounts for >95% of
total transport (see Fig. 2). At these concentrations, the
effect of irradiation on GSH transport revealed the response shown in
Fig. 4. Low doses of radiation (<36 Mrad)
resulted in an activation of GSH uptake that peaked at ~200% of the
rates measured in unirradiated samples. This rise was followed by a decay in transport at higher radiation doses (36-76 Mrad). At the
highest radiation doses (>76 Mrad), residual transport reached a
plateau ~20% of that in unirradiated samples. This ~20% residual fraction, insensitive to radiation, could again be attributed to
nonspecific effects and/or passive diffusion. To verify this point, the inhibition of residual transport of GSH at 50 mM was studied
in the presence of 5 mM diBSP. Uptake of GSH at all doses was inhibited
by diBSP (data not shown), resulting in inhibited residual values equal
to 24 ± 3% (mean ± SE, n = 3)
at the highest radiation dose.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Inactivation of low-affinity transport of GSH. Residual transport at 25 and 50 mM GSH from 4 (25 mM) and 7 (50 mM) independent preparations
were pooled. Mean values ( ), weighted by SEs (bars), were fitted
(solid line) with difference of 2 monoexponential functions plus a
constant value of 24% (see text). Broken lines represent resultant 2 exponentials obtained by fitting inactivation data (see target sizes in
text).
|
|
The data were therefore analyzed by fitting them with the difference of
two exponentials plus a constant equal to 24% (Fig. 4). This analysis
is based on a model of GSH transport that implies mediation of a
smaller transport unit with a larger structure that blocks or masks the
transporter. The estimated rate constant of the terminal (decaying)
exponential, defining the inactivation phase, yields the target size of
the transporter. The difference remaining after subtraction of the
terminal exponential plus the constant from the data defines a second
exponential that yields the target size of the larger "inhibitor"
or "regulator" protein. Fitting the mean values of
the data, pooled from 25 and 50 mM GSH transport experiments (weighted
by SEs shown in Fig. 4), gave a target size of 76 ± 11 (SE) kDa for
the low-affinity transporter and 141 ± 30 (SE) kDa for the
inhibitory or regulatory structure. In this analysis, the exponential
defining the inactivation of the low-affinity transporter extrapolates
back to ~600% of transport in unirradiated vesicles, indicating that
~80% of potential transport activity remains inhibited in
unirradiated samples. These estimated target sizes, obtained by fitting
the mean values of pooled data, were similar to estimates obtained by
fitting the data from individual experiments separately, calculated by
averaging all individual estimates representing the sizes obtained with
25 and 50 mM GSH (Table 2).
Radiation inactivation of cMOAT.
To compare the characteristics of inactivation and functional size of
cMOAT with those of the GSH transporter, we measured the inactivation
of ATP-dependent transport of LTC4
(a substrate with high affinity for cMOAT) in three additional
independent vesicle preparations. In all three preparations transport
of 1 µM LTC4 and in one
additional preparation transport of 5 µM
LTC4 were studied. Because cMOAT
is a 170-kDa protein, the range of radiation doses in these studies was
restricted to 0-35 Mrad. However, using 25 mM GSH, we confirmed
that the activation of low-affinity uptake of GSH occurred in these
vesicles at similar magnitude to those observed in the vesicles used to
study transport of GSH (data not shown).
As can be seen (Fig. 5), in direct contrast
to the low-affinity transport of GSH, irradiation resulted in a
monoexponential decay of LTC4
transport down to an irradiation-insensitive fraction equal to ~17%
of that in unirradiated controls. The functional size of cMOAT was
estimated by fitting an exponential plus a constant to the residual
ATP-dependent transport activities (fit shown in Fig. 5). Our analysis
yielded an average functional size of 302 ± 32 (SE) kDa, which
approximates a dimer of cMOAT.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Inactivation of ATP-dependent leukotriene (LT)
C4 uptake. Data represent residual
ATP-dependent transport of LTC4
(bars are SE) from duplicate determinations in 3 independent vesicle
preparations. Solid curve represents fit to data (see target size in
text). Note that maximal irradiation dose in these experiments was 35 Mrad.
|
|
 |
DISCUSSION |
The kinetics of GSH transport in CMV exhibit two distinct components:
1) a high-affinity Michaelis-Menten
element and 2) a low-affinity
sigmoidal element with n ~2 (Fig. 2;
Refs. 21 and 27). This feature made it possible to study the functional
size(s) of the GSH transport system by inactivation of transport at two distinct ranges of concentrations, reflecting essentially the activity
of each kinetic component separately. Radiation inactivation identifies
the mass of the molecular components required for GSH transport.
Nine independent CMV preparations were irradiated. The activity of
5'-nucleotidase and the transport of GSH and
LTC4 were assayed. Radiation
target sizes were obtained by analyses of the data from individual
experiments as well as from data pooled from multiple preparations. No
significant differences were observed between the results with the two
methods of analysis. The integral membrane enzyme 5'-nucleotidase
served as an internal control, yielding a target size in excellent
agreement with both published values from radiation analyses as well as
the known structure of the protein. These results validate both sample
treatment and radiation dosimetry in our studies.
The functional size of the high-affinity transporter was determined at
trace and 0.02 mM GSH concentrations. Radiation inactivation indicated
a single protein structure. The estimated mean target sizes obtained at
these two concentrations, after accounting for the noninhibitable
fraction, were indistinguishable, i.e., ~73 and ~69 kDa,
respectively. The low-affinity transporter was studied at 25 and 50 mM
GSH. In this case, radiation inactivation revealed a more complex
system, characterized by a rise and fall of transport. The estimated
size of the transporter from the pooled data at these high
concentrations of GSH was ~76 kDa, very close to that found for the
high-affinity transporter. The similarity of the functional sizes of
the high- and low-affinity components raises the possibility, but does
not prove, that the two kinetic components may be mediated by the same
protein.
The complex radiation inactivation pattern of the low-affinity
component indicated an initial "activation" of GSH transport at
low radiation doses. [Note that concomitant radiation
inactivation studies conducted on sinusoidal-enriched membrane vesicles
did not reveal such an activation pattern (unpublished
observations)]. Similar findings in other radiation
studies have been interpreted as the destruction of a larger inhibitory
or regulatory structure that masks the smaller transporter unit (12,
33, 34). With the combined data at 25 and 50 mM GSH, this larger
structure was estimated to be ~141 kDa. Extrapolation to zero dose of
radiation (y-axis intercept) of the exponential
that defines the inactivation of the transporter unit indicated that
>80% of potential GSH transport capacity in normal, unirradiated CMV
is suppressed by this larger structure. This outcome indicates that the
associated protein exerts a major inhibitory effect on the transport of
GSH in CMV. Thus it is conceivable that factors that determine the
secretion of GSH into bile may do so by transduction of signals to this regulator to enhance or decrease biliary GSH secretion. An alternative model might be considered, wherein GSH transport in CMV may be conducted by noncovalently associated homodimers (34). Because noncovalent bonds do not transmit radiation-induced damage (13), in
theory, irradiation could potentially liberate constituent, fully
active, monomeric transporters from homodimers. In this model, the
homodimers would be less active in low-affinity transport of GSH,
compared with monomers, which would be fully active. However, monomers
are not released from many different irradiated homo-oligomers (Kempner, unpublished observations), making this homodimeric model very
unlikely. The above two models differ in the characterization of the
GSH transport system as a hetero-oligomer (former) vs. a homo-oligomer
(latter). However, the radiation inactivation technique cannot
discriminate between such detailed molecular characteristics in a
membrane transport system in situ.
A transporter involved in the excretion of certain organic anions at
the canalicular domain of hepatocytes has been characterized (15, 29).
This 170-kDa protein termed cMOAT or MRP-2 is an ATP binding cassette
(ABC) transporter that mediates the flux of certain anionic conjugates
of GSH into bile (15, 29). Based on indirect evidence (see the
introduction), cMOAT would appear to be the principal canalicular
transporter for GSH (15, 29). However, many features of GSH transport
are inconsistent with those of cMOAT, as enumerated elsewhere (21). For
example, protein kinase C (PKC) activation enhances the secretion of
organic anions into bile (31) but inhibits biliary GSH secretion (21,
30). On the other hand, the reverse effect was observed
with PKC inhibitors (21, 30). These contrasting results of PKC
modulation could represent opposite effects on two distinct
transporters or two distinct functions of the same transporter. In both
cases GSH efflux could be mediated by cMOAT in an ATP-independent
manner (driven by electrogenic or electrochemical forces) and
functionally regulated in a manner distinct from ATP-driven organic
anion transport.
To investigate the relationship between GSH transport and cMOAT in our
model, we studied the inactivation of
LTC4 transport to compare the
functional sizes of the GSH transport system and cMOAT. Our estimate of
the functional size of cMOAT from the analysis of our data is ~302
kDa, approximating a dimer of 170 kDa (6, 29). Thus the minimal
functional size required for GSH transport in CMV, i.e., 70-76
kDa, is distinctly different from that of cMOAT. These findings
indicate that the canalicular GSH transporter is distinct from cMOAT.
However, because radiation inactivation cannot identify the details of
the molecular components of the GSH transport system, it is possible
that the larger regulatory protein associated with low-affinity GSH
transport may be cMOAT.
Two other possibilities, although less likely, cannot be unequivocally
ruled out at this time. First low-affinity ATP-independent transport of
GSH may be mediated by one of the two transmembrane domains of cMOAT.
As is a characteristic of many ABC proteins, cMOAT is a single
polypeptide containing two asymmetrical transmembrane domains and two
nucleotide binding domains. The functional size of the GSH transporter
would be consistent with cMOAT only if either transmembrane domain of
cMOAT exists as an independent polypeptide (due to posttranslational
modifications) and functions individually in the canalicular membrane.
Second, canalicular plasma membrane fractions are invariably
contaminated by certain intracellular organelles, such as endoplasmic
reticulum. Whether or to what extent these may contribute to
high-capacity GSH transport in the CMV preparations remains unknown and
subject to future investigations. Thus the postirradiation activation
of uptake in the CMV may reflect the characteristics of a high-capacity GSH carrier localized in an intracellular organelle. However, this
possibility is unlikely because radiation inactivation of GSH transport
in simultaneously isolated sinusoidal plasma membrane vesicles
(unpublished data) with similar intracellular organelle distributions
as CMV resulted in a distinct target size for low-affinity transport
and did not exhibit an activation of high- or low-affinity GSH
transport. Furthermore, the parallel operation of ATP-dependent transport of dinitrophenyl-glutathione (DNPSG; another
substrate for cMOAT) has been found to stimulate GSH uptake in these
CMV preparations (9, 10), presumably by transstimulation of GSH uptake
by the rapidly accumulating intravesicular
DNPSG. Irrespective of the mechanism of this
phenomenon, it unequivocally demonstrates that the same membrane
vesicles contain cMOAT and the GSH transporter.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institutes of Health Grants
R01-A607467 and R37-DK-30312. Rat liver plasma membrane vesicles were
prepared by the Subcellular-Organelle Core of the University of
Southern California Research Center for Liver Disease (RCLD; P30-DK-48522). Analysis and fitting of data were performed by the
Kinetic and Mathematical Analysis-Modeling Services of LDRC.
 |
FOOTNOTES |
Address for reprint requests: M. Ookhtens, Univ. of Southern California
School of Medicine, 1333 San Pablo St., MMR-428, Los Angeles, CA 90033.
Received 6 August 1997; accepted in final form 15 January 1998.
 |
REFERENCES |
1.
Amici, A.,
M. Emanuelli,
N. Raffaelli,
S. Ruggieri,
and
G. Magni.
One-minute high-performance liquid chromatography assay for 5'-nucleotidase using a 20-mm reverse-phase column.
Anal. Biochem.
216:
171-175,
1994[Medline].
2.
Ballatori, N.,
and
W. J. Dutczak.
Identification and characterization of high and low affinity transport systems for reduced glutathione in liver cell canalicular membranes.
J. Biol. Chem.
269:
19731-19737,
1994[Abstract/Free Full Text].
3.
Ballatori, N.,
R. H. Moseley,
and
J. Boyer.
Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles.
J. Biol. Chem.
261:
6216-6221,
1986[Abstract/Free Full Text].
4.
Ballatori, N.,
and
A. T. Truong.
Glutathione as a primary osmotic driving force in hepatic bile formation.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G617-G624,
1992[Abstract/Free Full Text].
5.
Bossard, R.,
B. Stieger,
B. O'Neil,
G. Fricker,
and
P. J. Meier.
Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver.
J. Clin. Invest.
91:
2714-2720,
1993[Medline].
6.
Buchler, M.,
J. Konig,
R. Brom,
J. Kartenbeck,
H. Spring,
T. Horie,
and
K. Keppler.
cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMRP, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats.
J. Biol. Chem.
271:
15091-15098,
1996[Abstract/Free Full Text].
7.
Dutczak, W. J.,
and
N. Ballatori.
Transport of the glutathione-methylmercury complex across liver canalicular membranes on reduced glutathione carriers.
J. Biol. Chem.
269:
9746-9751,
1994[Abstract/Free Full Text].
8.
Fariss, M. W.,
and
D. J. Reed.
High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives.
Methods Enzymol.
143:
101-109,
1987[Medline].
9.
Fernandez-Checa, J. C.,
M. Ookhtens,
and
N. Kaplowitz.
Selective induction by phenobarbital of the electrogenic transport of glutathione and organic anions in rat liver canalicular membrane vesicles.
J. Biol. Chem.
268:
10836-10841,
1993[Abstract/Free Full Text].
10.
Fernandez-Checa, J. C.,
H. Takikawa,
T. Horie,
M. Ookhtens,
and
N. Kaplowitz.
Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats.
J. Biol. Chem.
267:
1667-1673,
1992[Abstract/Free Full Text].
11.
Hagen, T. M.,
and
D. P. Jones.
Transepithelial transport of glutathione in vascularly perfused small intestine of rat.
Am. J. Physiol.
252 (Gastrointest. Liver Physiol. 15):
G607-G613,
1987[Abstract/Free Full Text].
12.
Harmon, J. T.,
C. R. Kahn,
E. S. Kempner,
and
W. Schlegel.
Characterization of the insulin receptor in its membrane environment by radiation inactivation.
J. Biol. Chem.
255:
3412-3419,
1980[Free Full Text].
13.
Harmon, J. T.,
T. B. Nielsen,
and
E. S. Kempner.
Molecular weight determinations from radiation inactivation.
Methods Enzymol.
117:
65-94,
1985[Medline].
14.
Inoue, M.,
R. Kinne,
T. Tran,
and
I. M. Arias.
The mechanism of biliary secretion of reduced glutathione: analysis of transport process in isolated rat-liver canalicular vesicles.
Eur. J. Biochem.
134:
467-471,
1983[Abstract].
15.
Ito, K.,
H. Suzuki,
T. Hirohashi,
K. Kume,
T. Shimizu,
and
Y. Sugiyama.
Molecular-cloning of canalicular multispecific organic anion transporter defective in EHBR.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G16-G22,
1997[Abstract/Free Full Text].
16.
Kaplowitz, N.,
T. Y. Aw,
and
M. Ookhtens.
The regulation of hepatic glutathione.
Annu. Rev. Pharmacol. Toxicol.
25:
715-744,
1985[Medline].
17.
Kaplowitz, N.,
D. E. Eberle,
J. Petrini,
J. Touloukian,
M. C. Corvasce,
and
J. Kuhlenkamp.
Factors influencing the efflux of hepatic glutathione into bile in rats.
J. Pharmacol. Exp. Ther.
263:
964-970,
1983[Abstract].
18.
Kempner, E. S.
Molecular size determination of enzymes by radiation inactivation.
Adv. Enzymol. Relat. Areas Mol. Biol.
61:
107-147,
1988[Medline].
19.
Kempner, E. S.
Novel predictions from radiation target analysis.
Trends Biochem. Sci.
18:
236-239,
1993[Medline].
20.
Kempner, E. S.
The mathematics of radiation target analyses.
Bull. Math. Biol.
57:
883-898,
1995[Medline].
21.
Lu, S. C.,
J. Kuhlenkamp,
H. Wu,
W. Sun,
L. Stone,
and
N. Kaplowitz.
Progressive defect in biliary GSH secretion in streptozotocin-induced diabetic rats.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G374-G382,
1997[Abstract/Free Full Text].
22.
Meier, P. J.,
A. S. Meier-Abt,
C. Barrett,
and
J. L. Boyer.
Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. Evidence for an electrogenic canalicular organic anion carrier.
J. Biol. Chem.
259:
10614-10622,
1984[Abstract/Free Full Text].
23.
Meier, P. J.,
E. S. Sztul,
A. Reuben,
and
J. L. Boyer.
Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver.
J. Cell Biol.
98:
991-1000,
1984[Abstract].
24.
Meister, A.,
and
M. E. Anderson.
Glutathione.
Annu. Rev. Biochem.
52:
711-760,
1983[Medline].
25.
Ookhtens, M.,
and
T. Maddatu.
Mechanism of changes in hepatic sinusoidal and biliary glutathione efflux with age in rats.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G648-G656,
1991[Abstract/Free Full Text].
26.
Ookhtens, M.,
and
A. V. Mittur.
Developmental changes in plasma thiol-disulfide turnover in rats: a multicompartmental approach.
Am. J. Physiol.
267 (Regulatory Integrative Physiol. 36):
R415-R425,
1994[Abstract/Free Full Text].
27.
Ookhtens, M.,
and
A. V. Mittur.
Maturational increase in canalicular GSH efflux is due to a rising Vmax of the low-affinity transporter (Abstract).
Hepatology
22:
814,
1995[Medline].
28.
Ookhtens, M.,
A. V. Mittur,
and
N. E. Erhart.
Changes in plasma glutathione concentrations, turnover, and disposal in developing rats.
Am. J. Physiol.
266 (Regulatory Integrative Physiol. 35):
R979-R988,
1994[Abstract/Free Full Text].
29.
Paulusma, C. C.,
P. J. Bosma,
G. J. Zaman,
C. T. Bakker,
M. Otter,
G. L. Scheffer,
R. J. Scheper,
P. Borst,
and
R. P. Oude-Elferink.
Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene.
Science
271:
1126-1128,
1996[Abstract].
30.
Raiford, D. S.,
A. M. Sciuto,
and
M. C. Mitchell.
Effects of vasopressor hormones and modulators of protein kinase C on glutathione efflux from perfused rat liver.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G578-G584,
1991[Abstract/Free Full Text].
31.
Roelofsen, H.,
R. Ottenhoff,
R. P. Oude-Elferink,
and
P. L. Jansen.
Hepatocanalicular organic-anion transport is regulated by protein kinase C.
Biochem. J.
278:
637-641,
1991[Medline].
32.
SAAM, II USER'S GUIDE.
SAAM Institute, University of Washington, Seattle, WA, 1994.
33.
Verkman, A. S.,
K. Skorecki,
and
D. A. Ausiello.
Radiation inactivation of oligomeric enzyme systems: theoretical considerations.
Proc. Natl. Acad. Sci. USA
81:
150-154,
1984[Abstract].
34.
Vessey, D. A.,
and
E. S. Kempner.
In situ structural analysis of microsomal UDP-glucuronyltransferases by radiation inactivation.
J. Biol. Chem.
264:
6334-6338,
1989[Abstract/Free Full Text].
35.
Yi, J.,
S. Lu,
J. C. Fernandez-Checa,
and
N. Kaplowitz.
Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics.
J. Clin. Invest.
93:
1841-1845,
1994[Medline].
AJP Gastroint Liver Physiol 274(5):G923-G930