We assessed the impact of zonal factors on the hepatic reduced
glutathione (GSH) conjugation of ethacrynic acid (EA). Uptake of EA by
enriched periportal (PP) and perivenous (PV) rat hepatocytes was
characterized by both saturable
(Vmaxuptake = 3.4 ± 1.7 and
3.2 ± 0.8 nmol/min/mg protein and
Kmuptake = 51 ± 13 and 44 ± 15 µM) and nonsaturable (12 ± 5 and 12 ± 3 µl/min/mg
protein) components. Values for the overall GSH conjugation rates of EA
(200 µM) were similar among the zonal hepatocytes and resembled those
for the influx transport rates. In the absence of the hepatocyte
membrane, GSH conjugation in PV and PP hepatocyte cytosol was similar,
but a higher perivenous GSH conjugation activity toward EA (PV/PP of
2.4) that mirrored the higher PV/PP ratios of immunodetectable GSTs Ya
(1.7) and Yb2 (2.5) was found in cell lysates obtained by the
dual-digitonin-pulse perfusion technique. The GSH conjugation rates in
the subcellular fragments were, however, much greater than those
observed for intact hepatocytes. Efflux rates of the glutathione
conjugate EA-SG from zonal hepatocytes were similar, as were levels of
the immunodetectable multidrug-resistance protein 2/canalicular
multispecific organic anion transporter (Mrp2/cMoat) in the
100,000g pellets. The composite results suggest that the
GSTs responsible for EA metabolism are more abundant in the PV region,
albeit that the gradient of enzymatic activities is shallow. Despite
the existence of zonal metabolic activity, the overall GSH conjugation
rate of EA is homogeneous among cells because the reaction is rate
limited by uptake, which occurs evenly. Results on EA-SG efflux suggest
the acinar homogeneity in Mrp2/cMoat function for canalicular transport.
 |
Introduction |
Glutathione
(GSH) conjugation is an important detoxification pathway for
electrophiles, including some therapeutic agents, carcinogens, and
reactive metabolites. Several factors have an impact on the rates of
GSH conjugation in liver; these include the cellular uptake or in situ
formation of acceptor substrates of GSH, the propensity for spontaneous
GSH conjugation, the presence of the glutathione
S-transferases (GSTs), and cosubstrate (GSH) availability.
Once formed, the GSH-adduct is effluxed out of the hepatocyte by
members of the multidrug resistance protein (MRP) family found at
either the canalicular or basolateral membrane (Büchler et al.,
1996
; Hirohashi et al., 1998; Keppler et al., 1998; König et al.,
1999). These processes are exemplified by the GSH conjugation of
ethacrynic acid (EA) in vitro and in the perfused rat liver (Tirona and
Pang, 1999). Data derived from in vitro spontaneous and enzymatic GSH
conjugation and hepatocytic uptake of EA had been successfully
scaled-up to describe observations from perfused rat liver with use of
a physiologic, kinetic model that encompassed transport and
bimolecular, spontaneous (nonenzymatic) and enzymatic metabolism. The
data suggest that hepatic uptake rate limited the GSH conjugation of EA
at low input concentrations (<50 µM), whereas cellular metabolism
played an increasingly greater role at higher concentrations (~200
µM). As intracellular levels of GSH became depleted as a result of
consumption, cosubstrate availability invariably rate limited GSH conjugation.
There is, additionally, an increasing awareness that acinar
heterogeneity in sinusoidal uptake and metabolism affects hepatic drug
processing (Pang, 1995). Kwon and Morris (1997) demonstrated, in
theory, that the total hepatic elimination of drugs would be influenced
by zonal localization of transport and enzymatic activities. For GSH
conjugation that is a bimolecular reaction, there is the extra
consideration of the unevenness in cellular availability of the
cosubstrate that could further affect the overall hepatic removal
(Pang, 1995). There exists much evidence that the GSTs are more
concentrated in the perivenous (PV) than the periportal (PP) regions
(Redick et al., 1982). The rat liver GSTs that are constitutively
expressed and capable of metabolizing EA (Ploemen et al., 1991, 1993),
such as the 
(subunits 1, 2, and 8) and µ (subunits 3 and 4)
classes, are also more abundant in the PV zone (Sippel et al., 1996).
Other GSTs (the microsomal GSTs and those belonging to the 
class)
exhibit negligible activity toward EA (Ploemen et al., 1993), and these
are also constitutive to the PV region (Mainwaring et al., 1996). The
transport of EA into isolated rat hepatocytes was facilitated by a
sodium-independent transporter whose identity and zonal distribution
are currently unknown and by a nonsaturable (linear) system (Tirona and
Pang, 1999). It is conceivable that zonal uptake exists for EA because this zonated event occurs within PV hepatocytes for substrates such as
cysteine (Saiki et al., 1992), glutamate (Burger et al., 1989; Tan et
al., 1999), and -ketoglutarate (Moseley et al., 1992). Likewise,
predominance of immunodetectable sinusoidal transport proteins was
observed in PV hepatocytes for the rat glucose transporter 1 (GLUT1;
Tal et al., 1990), the rat organic cation transporter 1 (rOCT1;
Meyer-Wentrup et al., 1998), and the rat organic anion transporting
polypeptide 2 (oatp2; Kakyo et al., 1999). By contrast, acinar
homogeneity exists for the rat sodium-dependent taurocholate transporting polypeptide, ntcp (Stieger et al., 1994; Tan et al., 1999), and the organic anion transporting polypeptide from rat liver,
oatp1 (T. N. Abu-Zahra, A. W. Wolkoff, R. B. Xim, and K. S. Pang,
unpublished observations), in their uptake of substrates.
In this communication, we investigated the initial rates of uptake of
EA and explored the possible roles of acinar metabolism and transport
by isolated, enriched PP and PV rat hepatocytes. We further studied
accumulation of the formed GSH adduct of EA (EA-SG) because it is
recognized that GSH conjugates, including EA-SG, are product-inhibitors
of GSTs (Ploemen et al., 1990), and the efficiency in efflux of GSH
conjugates may play an important role in the cellular detoxification of
electrophiles by the GSTs. The transporters responsible for this
basolateral efflux of EA-SG in the rat have not been directly
identified but are likely orthologs of human MRP family proteins (Zaman
et al., 1997). However, considerably faster efflux of EA-SG occurs with
the canalicular multispecific organic anion transporter, cMoat or Mrp2
(Büchler et al., 1996, Evers et al., 1998), and this accounted
for the rapid appearance of EA-SG excretion in rat bile (Tirona and
Pang, 1999). It is unknown whether acinar heterogeneity exists for the
efflux processes in rat liver.
| |
Experimental Procedures |
Materials.
[14C]EA (specific activity, 15 mCi/mmol) was a kind gift from Dr. J. H. T. M. Ploemen
(TNO, Ziest, the Netherlands) and was purified by high-performance
liquid chromatography (HPLC) and solid-phase extraction (radiochemical
purity >98%). [3H]Sucrose (specific activity, 10 Ci/mmol) was obtained from NEN Life Sciences (Boston, MA). EA,
1-chloro-2,4-dinitrobenzene (CDNB), GSH, and oxidized GSH (GSSG) were
purchased from Sigma Chemical Co. (St. Louis, MO). EA-SG was
synthesized as described previously (Tirona and Pang, 1999). Digitonin
was obtained from Fluka Chemie (Buchs, Switzerland). Collagenase was
purchased from Boehringer-Mannheim (Oakville, Ontario, Canada).
Antisera raised against rat GST Ya (rGST 1-1) and Yb2 (rGST 4-4) were
obtained from Biotrin International (Dublin, Ireland). Polyclonal
antibodies against rat Mrp2/cMoat (EAG15; Büchler et al., 1996)
and monoclonal antibodies against rat cytochrome P-450 1A (CYP1A)
isozymes (MAb 1-7-1) were generously provided by Drs. D. Keppler
(Deutsches Krebsforschungszentrum, Heidelberg, Germany) and H. V. Gelboin (National Institutes of Health, Bethesda, MD), respectively.
All other reagents were of the highest available grade.
Isolation of PP and PV Rat Hepatocytes.
Enriched PP and PV
hepatocytes from male Sprague-Dawley rats (275-325 g; Charles River
Canada, St. Constant, Quebec, Canada) were harvested by the
digitonin/collagenase perfusion method according to Lindros and
Pentïlla (1985), with slight modifications as detailed by Tan
et al. (1999). Hepatocyte viability (>90%) was assessed by Trypan
blue exclusion. Zonal enrichment was routinely estimated by monitoring
the activities of alanine aminotransferase (ALT) with a commercially
available kit (Sigma) and of glutamine synthetase (GS) by a standard UV
method (Tan et al., 1999). Protein was determined by the method of
Lowry et al. (1951). Several low-speed centrifugations (50g)
during the isolation procedure separated most of the nonparenchymal
cells from hepatocytes; it was surmised that low levels of
contamination persisted.
Uptake of EA by PP and PV Rat Hepatocytes.
The zonal
hepatocytes were suspended in Krebs-Henseleit bicarbonate buffer (pH
7.4) supplemented with 5 mM glucose and 1 mM HEPES and were
preconditioned for 10 min at 37°C. A mixture of [14C]EA, [3H]sucrose, and unlabeled EA was
added to the cell suspension to result in final EA concentrations of 1 to 800 µM and ~1.67 × 106 cells/ml. Samples were
retrieved at 15- to 20-s intervals after admixture and were rapidly
centrifuged through a layer of silicon oil as described previously (Tan
et al., 1999). Cellular radioactivity was determined by liquid
scintillation spectrometry (LSC, model 5801; Beckman Canada,
Mississauga, Ontario, Canada), after correction of the adhered water
layer defined by [3H]sucrose (Tan et al., 1999). Because
the initial cellular accumulation of EA was linear over 80 s, the
rate of uptake, vuptake, was estimated as
the slope on regression of the accumulated amount-versus-time data. The
kinetics of EA uptake were analyzed by fitting
vuptake against the initial substrate
concentration [EA] with use of the following equation (eq. 1, after
elimination of other possibilities of single and multiple saturable
systems) and an appropriate weighting scheme with a least-squares
fitting routine with the software SCIENTIST (Micromath Scientific
Software, Salt Lake City, UT):
![v<SUB><IT>uptake</IT></SUB>=<FR><NU>V<SUP><IT>uptake</IT></SUP><SUB><UP>max</UP></SUB>[EA]</NU><DE>K<SUP><IT>uptake</IT></SUP><SUB>m</SUB>+[EA]</DE></FR>+P<SUB><IT>diff</IT></SUB>[EA]](/content/vol291/issue3/fulltext/1210/img001.gif)
|
(1)
|
where Vmaxuptake and
Kmuptake are the maximum uptake rate
and the Michaelis-Menten constant for the saturable process, and
Pdiff is the uptake clearance for the
linear component.
Metabolism of EA by PP and PV Rat Hepatocytes.
We chose to
study EA at the concentration of 200 µM because GSH conjugation of
the drug would be influenced by both cellular uptake and enzymatic
activity (Tirona and Pang, 1999). Hence, inherent acinar differences of
these factors would be more apparent. EA, dissolved in physiological
saline solution, was added to the hepatocyte suspension to achieve an
initial concentration of 200 µM with ~1.67 × 106
cells/ml. At 1- to 5-min intervals throughout the 20-min incubation experiment conducted at 37°C, samples (500 µl) were retrieved and
placed into 1.5-ml microcentrifuge tubes containing 70 µl of 70%
perchloric acid. A separate sample (700 µl) was overlaid onto 250 µl of 1-bromododecane and centrifuged (Biofuge pico; Heraeus
Instruments, Germany) for 10 s. An aliquot (500 µl) of the
resulting supernatant was similarly placed into 70% perchloric acid to
assay for contents in the extracellular space of the incubation mixture. The acidified samples were immediately mixed and stored at
70°C until analysis.
Analysis.
For quantitation of EA and EA-SG, each acidified
sample (total incubation mixture and extracellular medium) was further
combined with 200 µl of 1.2 mM 4-(2,4-dichlorophenoxy)-butyric acid
(internal standard). After centrifugation, 100 µl of the supernatant
was analyzed by HPLC according to a previously developed procedure (Tirona and Pang, 1999). Standard curves, prepared from solutions of
known concentrations of EA (50-250 µM) and EA-SG (10-250 µM), were constructed in a similar fashion.
For the analysis of GSH and GSSG, 500 µl of the total suspension
(cell and extracellular medium) and 500 µl of the extracellular medium were added to microcentrifuge tubes containing 100 µl of 70%
perchloric acid and 50 µl of 15 mM bathophenanthroline-disulfonic acid. After centrifugation, the supernatant was immediately analyzed for GSH and GSSG with use of the derivatization and HPLC method of
Fariss and Reed (1987). The intracellular concentrations of EA, EA-SG,
and GSH/GSSG were estimated as the difference between the
concentrations in the total suspension and the extracellular medium.
Acid-precipitated hepatocytes were further analyzed for the GSH-protein
mixed disulfide content with the same HPLC method (Fariss and Reed,
1987).
GST Activity of PP and PV Hepatocyte Cytosols.
Cytosol was
obtained by homogenization of the zonal hepatocytes (Ultra-Turrax T25
homogenizer; Janke & Kunkel, Staufen im Briesgau, Germany), and the
resultant supernatant fractions were sequentially centrifuged at
9,000g and 100,000g at 4°C. Cytosolic GST
activity toward EA was determined within the linear protein concentration range by the spectrophotometric method of Satoh (1995) as
previously described (Tirona and Pang, 1999) with 200 µM EA and 5 mM
GSH, at 37°C and pH 7.2. Cytosolic GST activity toward CDNB was
determined by standard UV methods with 1 mM CDNB and 1 mM GSH, at
25°C and pH 6.5 (Habig et al., 1974). The GST-catalyzed GSH
conjugation activities toward EA and CDNB were obtained after correction of the (total) cytosolic GSH conjugation rates for the
spontaneous reaction rates (in absence of cytosol) and normalization to
the protein contents.
PP and PV Cell Lysate.
Preparation of the cell lysates from
the most proximal and distal hepatocytes along the sinusoidal plate was
performed according to the dual-digitonin-pulse perfusion method of
Quistorff and Grunnet (1987), with modifications. Paired zonal (PP and
PV) lysates were prepared from the same liver. Rat livers were perfused
with Hanks' buffer (pH 7.2) containing 10 mM HEPES, 0.5 mM EGTA, 4.2 mM NaHCO3, and 5 mM glucose (perfusion buffer) pregassed
with 95% O2/5% CO2 at a flow rate of 30 ml/min into the portal vein in a prograde fashion. After 10 min, the
flow rate was reduced to 12 ml/min, and the direction of flow was
reversed (retrograde perfusion into the hepatic vein). After
stabilization of the liver for 1 min, the perfusion medium was changed
to the digitonin solution (3.25 mM digitonin, 150 mM NaCl, 6.7 mM KCl,
and 50 mM HEPES) for perfusion at 6 ml/min until a spotted destruction
pattern was observed on the liver surface (35 ± 11 s) for
destruction of the PV region. The flow was then reverted to prograde
flow, and perfusion buffer was used for perfusion at a rate of 20 ml/min. The eluate (PV lysate) was collected over 30 s. For
continued preparation of the PP lysate, the direction of flow was
reversed. Rat livers were perfused with perfusion buffer at the flow
rate of 20 ml/min into the hepatic vein in a retrograde fashion for 2.5 min. Then, the flow rate was reduced to 12 ml/min for 1 min. Next, the
digitonin solution was infused at 6 ml/min until a circular destruction
pattern appeared (26 ± 8 s) for destruction of the PP
region. Subsequently, the eluate (PP lysate) was collected for 30 s. PP and PV eluates were centrifuged at 100,000g for 60 min
at 4°C, and the resultant supernatants were stored at 70°C.
Kinetic Modeling of EA Disposition by PP and PV Hepatocytes.
A kinetic model, whose scheme is shown in Fig.
1, was used for analysis of the
time-dependent disposition of EA in PP and PV hepatocytes. The
saturable uptake (Kmuptake and
Vmaxuptake) and the bidirectional
(uptake and efflux) linear clearance (Pdiff) parameters, obtained from uptake experiments (Fig.
2), were used to denote transmembrane
transport. The spontaneous and GST-catalyzed GSH conjugations of EA
were described by a second-order (spontaneous) reaction and a single
enzyme-catalyzed, rapid-equilibrium, random sequential order scheme as
reported previously (Tirona and Pang, 1999). The second-order constant
(k2) for the spontaneous reaction and the
apparent Michaelis-Menten constants for GSH
(KmGSH) and EA
(KmEA) for the enzymatic reactions were
obtained from the previous in vitro studies (Tirona and Pang, 1999).
The maximal enzymatic conjugation rate
(Vmaxmetab) was estimated by the
fitting procedure.
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Model for GSH conjugation of EA in isolated
hepatocytes. Cellular EA uptake is represented by saturable and linear
(bidirectional) processes. GSH conjugation is modeled using
second-order spontaneous and bimolecular enzymatic reactions. Net EA-SG
efflux into the extracellular medium and into a sequestered vesicular
space are described by linear clearances. See text for details.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Concentration-dependent uptake of
[14C]EA by isolated rat PP (A) and PV (B) hepatocytes.
The solid line was obtained by fitting data with eq. 1 where the dotted
line indicates the saturable component and the dashed line represents
the nonsaturable component for EA uptake (mean ± S.D.,
n = 4).
|
|
EA-SG formed within isolated hepatocytes was assumed to undergo net
efflux into the extracellular medium and transport into intracellular
sequestration vesicles (see Discussion). These linear clearance processes, denoted by the terms
CLeffluxEA-SG and
CLvesEA-SG, respectively, were
estimated by fitting. Because control experiments indicated a lack of
net change (over 20 min) in intracellular hepatocyte GSH (Figs.
3A and 4A)
and because GSH synthesis is expected to be insignificant in the
absence of GSH precursors under the experimental conditions,
intracellular GSH kinetics were simplified; the loss of GSH was due
only to conjugation. However, there was a lack of mass balance because
the amount of GSH consumed was less than the total amount of EA-SG
formed. The intracellular GSH concentrations were scaled-up to allow
for mass balance for the purposes of fitting; the GSH data were
converted to concentration terms by assuming 10 µl of cell volume/ml
of suspension. The mean data of the PP and PV hepatocyte experiments were simultaneously fitted with the model equations with use of a
nonlinear least-squares procedure (SCIENTIST) and appropriate weighting
schemes. The fitted parameter values, together with the assigned
parameters, are shown (see Table 3).
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
GSH conjugation of EA (200 µM) by isolated PP rat
hepatocytes. GSH (A), EA (B), and EA-SG (C) contents were determined by
HPLC (mean ± S.D., n = 6) in the total
suspension, and extracellular and cellular spaces. The solid, dotted,
and dashed lines represent the fits to the kinetic model for
extracellular, intracellular, and total species, except for A, where
fits to the intracellular GSH are shown. The inset in A shows the GSH
contents in control (saline-treated) PP hepatocytes (n = 4).
|
|
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
GSH conjugation of EA (200 µM) by isolated PV rat
hepatocytes. GSH (A), EA (B), and EA-SG (C) contents were determined by
HPLC (mean ± S.D., n = 6) in the total
suspension, and extracellular and cellular spaces. The solid, dotted,
and dashed lines represent the fits to the kinetic model for
extracellular, intracellular and total species, except for A, where
fits to the intracellular GSH are shown. The inset in A shows the GSH
contents in control (saline-treated) PV hepatocytes (n = 4).
|
|
The following mass balance rate equations are used in the model (Fig.
1).
The equations describing the extracellular space (EC) for EA and EA-SG
are
![<FR><NU>d[EA]<SUB>EC</SUB></NU><DE>dt</DE></FR>=<FENCE><UP>−</UP><FR><NU>V<SUP><IT>uptake</IT></SUP><SUB><UP>max</UP></SUB>[EA]<SUB>EC</SUB></NU><DE>K<SUP><IT>uptake</IT></SUP><SUB>m</SUB>+[EA]<SUB>EC</SUB></DE></FR>−P<SUB><IT>diff</IT></SUB>[EA]<SUB>EC</SUB>+P<SUB><IT>diff</IT></SUB>[EA]<SUB>C</SUB></FENCE>/V<SUB>EC</SUB>](/content/vol291/issue3/fulltext/1210/img002.gif)
|
(2)
|
![<FR><NU>d[EA-SG]<SUB>EC</SUB></NU><DE>dt</DE></FR>=(CL<SUP>EA-SG</SUP><SUB><IT>efflux</IT></SUB>[EA-SG]<SUB>C</SUB>)/V<SUB>EC</SUB>](/content/vol291/issue3/fulltext/1210/img003.gif)
|
(3)
|
whereas the equations describing the cellular space (C) are
![<FR><NU>d[EA]<SUB>C</SUB></NU><DE>dt</DE></FR>=<FENCE><FR><NU>V<SUP><IT>uptake</IT></SUP><SUB><UP>max</UP></SUB>[EA]<SUB>EC</SUB></NU><DE>K<SUP><IT>uptake</IT></SUP><SUB>m</SUB>+[EA]<SUB>EC</SUB></DE></FR>+P<SUB><IT>diff</IT></SUB>[EA]<SUB>EC</SUB>−P<SUB><IT>diff</IT></SUB>[EA]<SUB>C</SUB>−k<SUB>2</SUB>[EA]<SUB>C</SUB>[GSH]<SUB>C</SUB>V<SUB>C</SUB></FENCE>](/content/vol291/issue3/fulltext/1210/img004.gif)
|
(4)
|
![<FR><NU>d[EA-SG]<SUB>C</SUB></NU><DE>dt</DE></FR>=(k<SUB>2</SUB>[EA]<SUB>C</SUB>[GSH]<SUB>C</SUB>V<SUB>C</SUB>](/content/vol291/issue3/fulltext/1210/img006.gif)
|
(5)
|
![<FR><NU>d[GSH]<SUB>C</SUB></NU><DE>dt</DE></FR>=<FENCE><UP>−</UP>k<SUB>2</SUB>[EA]<SUB>C</SUB>[GSH]<SUB>C</SUB>V<SUB>C</SUB>−<FR><NU>V<SUP><IT>metab</IT></SUP><SUB><UP>max</UP></SUB>[EA]<SUB>C</SUB>[GSH]<SUB>C</SUB></NU><DE>K<SUP>GSH</SUP><SUB>m</SUB>K<SUP>EA</SUP><SUB>m</SUB>+K<SUP>EA</SUP><SUB>m</SUB>[GSH]<SUB>C</SUB>+K<SUP>GSH</SUP><SUB>m</SUB>[EA]<SUB>C</SUB>+[EA]<SUB>C</SUB>[GSH]<SUB>C</SUB></DE></FR></FENCE>/V<SUB>C</SUB>](/content/vol291/issue3/fulltext/1210/img009.gif)
|
(6)
|
and the equation which describes the accumulation of EA-SG
within the vesicular sequestration space (V) is
![<FR><NU>dEA-SG<SUB>V</SUB></NU><DE>dt</DE></FR>=CL<SUP>EA-SG</SUP><SUB><IT>ves</IT></SUB>[EA-SG]<SUB>C</SUB>](/content/vol291/issue3/fulltext/1210/img010.gif)
|
(7)
|
where [EA], [EA-SG], and [GSH] are the concentrations of
EA, EA-SG, and GSH in each of the compartments (subscripts EC, C, and V
for extracellular, cellular, and vesicular spaces, respectively). Total
intracellular EA-SG is represented by the sum of cellular and vesicular contents.
Immunoblot Analysis.
Cytosols, lysates (5 µg of protein
containing the GSTs), and 100,000g pellets (10 µg of
protein containing Mrp2 and the marker protein CYP1A2) derived from PP
and PV hepatocytes were used for analysis. The immunoreactive proteins
were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on 12, 9, and 7.5% gels for the GSTs, CYP1A2, and Mrp2, respectively, using
the MiniProtean II system (Bio-Rad, Mississauga, Ontario, Canada).
Protein was transferred onto nitrocellulose membranes (Hybond ECL;
Amersham, Oakville, Ontario, Canada) with a semidry transfer unit
(Bio-Rad). Subsequently, the membranes were blocked with 10% nonfat
milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h at
room temperature. After washing with TBST, membranes were incubated
with primary antibody (anti-GST Ya or Yb2 at 1:50,000 dilution, MAb
1-7-1 at 1:20,000 dilution, and EAG15 at 1:40,000 dilution) in TBST
overnight at 4°C. After washing with TBST, the membranes were
incubated with horseradish peroxidase-linked anti-rabbit (GST and Mrp2) or anti-mouse immunoglobulins (CYP1A2; Amersham or Bio-Rad) at 1:20,000
dilution in TBST for 2 h at room temperature. Detection was
performed using enhanced chemiluminescence (ECL; Amersham), and
membranes were visualized on Hyperfilm (Amersham). For the semiquantitation of the GSTs, CYP1A2, and Mrp2, the films were scanned
(Umax Astra 1200S), and band intensities were integrated using the NIH
Image software (developed at the National Institutes of Health and
available on the Internet at http://rsb.info.nih.gov/nih-image/).
Statistical Analysis.
All data are presented as mean ± S.D. Unpaired and paired Student's t tests were used where
appropriate; the level of significance was set at .05.
| |
Results |
Biochemical Characterization of PP and PV Hepatocytes and
Lysates.
The activities of PP and PV marker enzymes (ALT and GS,
respectively) are summarized in Table 1.
Significant differences were observed in marker enzyme activities
indicating the attainment of the zonal enrichment of PP and PV
hepatocytes (p < .05) and PP and PV lysates
(p < .05). The PP/PV activity ratios for ALT were
higher for lysate (7.5) than for hepatocyte cytosol (1.8), confirming
the steeper and decreasing (portal to venous) acinar gradient in enzyme
content. The PP/PV activity ratios for GS were similar among lysates
and hepatocyte cytosols (0.025 and 0.029, respectively), and these
results are consistent with the confined localization of this enzyme to
the terminal two or three hepatocytes in the PV region. These
observations established the validity of the preparation on the
enrichment of PP and PV hepatocytes or lysates.
Concentration-Dependent Uptake of EA by Zonal Hepatocytes.
EA
uptake kinetics were similar among PP and PV hepatocytes (Fig. 2). The
uptake parameter estimated on fitting of the data to eq. 1 furnished
similar Kmuptake (51 ± 13 and
44 ± 15 µM) and
Vmaxuptake (3.4 ± 1.7 and
3.2 ± 0.8 nmol/min/mg protein) values for the saturable uptake of
EA for PP and PV hepatocytes (p > .05, n = 4);
comparable values were also obtained for the nonsaturable component,
Pdiff (12 ± 5 and 12 ± 3 µl/min/mg protein) for PP and PV hepatocytes (p > .05, n = 4).
Cellular GSH Concentration and GSH Conjugation Rates of EA by PP
and PV Hepatocytes.
Control (saline-treated) PP and PV hepatocytes
(n = 4) retained their initial intracellular GSH
contents for at least 20 min, and the extracellular GSH levels remained
constant throughout the incubation period (Figs. 3A and 4A, insets).
GSSG, found mainly in the extracellular space, remained virtually
constant during the incubation and accounted for approximately 10 to
15% of the total GSH equivalents in the system (data not shown).
The addition of EA (200 µM) to PP and PV hepatocyte suspensions
(n = 12) greatly reduced GSH levels, which in turn
affected the rate of GSH conjugation and disappearance of EA (Figs. 3A and 4A). Although the extracellular GSH levels remained constant throughout the incubation period in treated hepatocytes and were similar to those of the controls, the GSH within PP and PV hepatocytes was rapidly depleted within the first 5 min after EA treatment (Figs.
3A and 4A). The pattern of depletion rate was, however, independent of
the acinar origin of the hepatocytes.
EA disappeared at similar rates in total suspension and the
extracellular medium of the incubation system with PP and PV
hepatocytes (Figs. 3B and 4B). Cellular accumulation of EA was low in
either PP or PV hepatocytes. The loss of EA was completely accounted for by the formation of EA-SG, and no other sequential metabolite (cysteinyl-glycine, cysteine and N-acetyl-cysteine adducts)
was detected. Essentially, mass balance was conserved by EA and EA-SG throughout the 20-min incubation period; the dose recovery was 100 ± 5 and 100 ± 6% for the PP and PV hepatocyte incubation
systems, respectively.
Both PP and PV hepatocytes produced EA-SG at equivalent rates (Figs. 3C
and 4C). Because rapid efflux of EA-SG occurred on its formation within
cells, the formation rate of EA-SG was more appropriately estimated by
viewing the early-in-time data for total concentration in the
incubation system before significant depletion of cellular GSH
occurred. The initial formation rates (roughly estimated from upslope
of the data between 0 to 3 min) of 6.2 ± 0.9 and 6.9 ± 1.1 nmol/min/mg protein, respectively, were comparable (p > .05) and were similar to those observed for uptake (5.4 ± 1.5 and 5.2 ± 0.9 nmol/min/mg protein, respectively; Table
2) at 200 µM. This suggests that
glutathione conjugation occurred very rapidly on cellular uptake of EA.
The EA-SG formation rate rapidly approached its asymptotic values by 10 min (Figs. 3C and 4C) due to the depletion of GSH (Figs. 3A and 4A).
The EA-SG formed within the zonally enriched hepatocytes rapidly
escaped into the extracellular media in a monoexponential fashion.
After 15 min, EA-SG remained sequestered within the hepatocyte with incubation time, and the net efflux of cellular EA-SG approached zero.
Because the efflux rate equaled the rate of accumulation in
extracellular medium, the initial EA-SG efflux rate was estimated from
the early-in-time data for the extracellular medium (1.5-5 min,
excluding zero time due to the short lag-time involved with the
cellular formation/cellular distribution/appearance of EA-SG in
medium). These were similar among the zonal hepatocytes (2.2 ± 0.2 and 2.3 ± 0.3 nmol/min/mg protein, respectively, p > .05), and the accumulation of EA-SG in extracellular medium
approached their corresponding asymptotic values by 15 min. Although
the rates of initial efflux of EA-SG were considerably slower than those of formation, the accumulation of intracellular EA-SG was short-lived because EA-SG formation ceased by 7.5 min due to the almost
complete depletion of GSH (Figs. 3A and 4A).
It was noted that the total formation of the EA-SG (~25 nmol/mg
protein) at the end of the experiment exceeded the total loss of
cellular GSH (from ~15-17 nmol/mg protein to almost zero). Because
the summed loss of EA equated with the production of EA-SG, it is
unlikely that experimental errors occurred in the quantitation by HPLC.
GSH synthesis within the hepatocytes could account for the difference
because the rate of GSH synthesis by hepatocytes has been estimated to
be ~0.4 nmol/min/106 cells (Lu et al., 1991). This
possibility, however, is remote due to the lack of amino acid
precursors in the suspension medium and the lack of net change in GSH
in control hepatocytes (Figs. 3A and 4A, insets). Furthermore, total
EA-SG formation was discontinued by 10 min, an observation that
contradicts the sustained GSH synthesis. The levels of GSH-protein
mixed disulfides, measured in a separate group of hepatocytes,
accounted for only ~1% of the total GSH content. At this time, we
have no explanation for this discrepancy.
Fitting of Data to Kinetic Model.
Reasonable fits to the data
were obtained on scale-up of intracellular GSH to provide for
mass-balance (Figs. 3 and 4). Values for the fitted
Vmaxmetab,
CLeffluxEA-SG, and
CLvesEA-SG (Table 3) revealed
that the processes governing the enzymatic formation of EA-SG and its
transport were all similar among the zonal hepatocytes. We further
tried out other kinetic models that do not include an intracellular
sequestration space; all failed to provide adequate fits to the
observations because these predicted complete intracellular depletion
of EA-SG by the end of the incubation experiment (fits not shown).
The fits showed that the initial rates of EA-SG formation were somewhat
underestimated by the model (Figs. 3C and 4C) when parameters observed
for EA transport were assigned with values obtained from the uptake
experiments. These values, based on [14C]EA influx in
rapid uptake studies (Fig. 2, Table 2), predicted a slightly lower
initial rate of EA uptake that resulted in a more gradual decline in EA
disappearance and lowered formation of EA-SG during the first 3 min of
incubation (Figs. 3 and 4). A systematic trend occurred with the
fitting of the cellular and extracellular EA-SG data (Figs. 3C and 4C)
and might have been the consequence of lack of modeling of the
time-dependent decrease in
CLeffluxEA-SG and concomitant
increase in CLvesEA-SG with
progressive internalization of Mrp2 during the time-span of the
experiment. Moreover, transport of EA-SG from cell to extracellular medium was described only by a net efflux clearance parameter (CLeffluxEA-SG) and failed to
incorporate bidirectional movement.
The present data for efflux of EA-SG from hepatocytes were further
scaled-up with the scaling factor (/
; where is 1.25 × 108 cells/g liver and is 1 × 106
cells/mg protein) and compared with that obtained from the whole organ
(Tirona and Pang, 1999). The calculation was based on the assumption
that the total efflux clearance for EA-SG in hepatocytes equaled the
sum of CLeffluxEA-SG and
CLvesEA-SG (~1.2 µl/min/mg),
yielding an efflux clearance of 1.5 ml/min/10 g liver for whole liver.
The estimated value was similar to the sum of sinusoidal and biliary
clearance for EA-SG (1.1 ml/min/10 g liver) in perfused rat liver
studies (Tirona and Pang, 1999).
The Vmaxmetab estimates (~35
nmol/min/mg) obtained with model fitting (Table 3) with the hepatocyte
experiments were much lower than that observed for the GST activity in
vitro (in cytosol) toward EA-SG formation from 200 µM EA and 5 mM GSH
at physiological conditions (~200 nmol/min/mg, Table 2). This big
discrepancy was the result of the very poor reliability of
Vmaxmetab because only one
concentration was studied and saturation of metabolism might not have
been attained under the experimental condition. These led to the large
standard deviation of the estimate (Table
3).
GST Activities in Cytosolic Fractions of PP and PV Hepatocytes and
Lysates.
The in vitro GST activities derived from cytosols of PP
and PV hepatocytes and PP and PV lysates toward EA are summarized in
Fig. 5A. No difference in GST activity
was observed among zonally enriched hepatocytes, whereas a 2.4-fold
greater activity (p < .05) was observed for the PV
lysate compared with the PP lysate. The GST activities toward EA
mirrored those toward CDNB (Fig. 5B). No difference in cytosolic GST
activity was seen in zonal hepatocytes toward CDNB, whereas a 1.9-fold
difference (p < .05) was observed between PV and PP
lysates.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
GST activity toward EA (A) and CDNB (B) in
cytosols prepared from isolated PP and PV rat hepatocytes (mean ± S.D.; n = 4 for PP and n = 4 for PV
hepatocyte preparations from eight rat livers) and in dual
digitonin-pulse perfusion lysates (paired PP and PV samples from four
rat livers). *, PV activity is significantly different (p < .05, paired Student's t test) from PP activity.
|
|
GSTs in PP and PV Hepatocytes and Lysates.
The levels of two
constitutive rat liver GSTs (Ya and Yb2) were found to be similar for
the cytosolic fractions of PP and PV hepatocytes (Fig.
6) when these were assessed by SDS-PAGE
and densitometry. However, the GST Ya and Yb2 proteins in PV
lysate were 1.7 and 2.5 times those of the PP lysates (Fig.
7).
View larger version (39K):
[in this window]
[in a new window]
|
Fig. 6.
Immunoblot analysis of GST Ya and Yb2 from the
cytosols derived from isolated PP and PV hepatocytes (n = 4 PP and n = 4 PV hepatocyte preparations from
eight rat livers). Lanes 1 to 4 and 5 to 8, respectively, denote
samples from PP and PV hepatocyte cytosols. Proteins (5 µg) were
separated by SDS-PAGE on 12% gels, transferred onto nitrocellulose,
and probed with antisera raised against rat GSTs (Biotrin). Detection
was performed using the ECL method, and immunoreactive bands were
analyzed by densitometry (mean ± S.D.).
|
|
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 7.
Immunoblot analysis of GST Ya and Yb2 from PP and PV
dual-digitonin-pulse perfusion lysates (paired PP and PV samples from
n = 4 rat livers, 1 to 4) Lanes marked 1, 2, 3, and 4 denote paired PP and PV samples obtained from that particular rat
liver. Proteins (5 µg) were separated by SDS-PAGE on 12% gels,
transferred onto nitrocellulose, and probed with antisera raised
against rat GSTs (Biotrin). Detection was performed using the ECL
method, and immunoreactive bands were analyzed by densitometry
(mean ± S.D.). *, PV absorbance is significantly different
(p < .05, paired Student's t test) from
that of PP.
|
|
Mrp2 in PP and PV Hepatocytes.
Levels of immunoreactive
Mrp2 in crude membranes obtained from 100,000g pellets of
homogenized PP and PV hepatocytes are shown in Fig.
8. PP and PV hepatocytes contained
similar amounts of Mrp2 protein. By contrast, the same membrane
fractions contained a highly variable and 3- to 4-fold enrichment of
constitutively expressed CYP1A2 in PV hepatocytes (p < .5), as anticipated for the marker protein.
View larger version (39K):
[in this window]
[in a new window]
|
Fig. 8.
Immunoblot analysis of Mrp2/cMoat and CYP1A2 (a
marker protein for PV abundance) from crude membranes
(100,000g pellets) of isolated PP and PV hepatocytes
(n = 4 PP and n = 4 PV hepatocyte
preparations from eight rat livers). Lanes 1 to 4 and 5 to 8 indicate
samples from PP and PV hepatocyte membrane fractions, respectively, of
the various rat livers. Proteins (10 µg) were separated by SDS-PAGE
on 7.5% (MRP2) and 9% (CYP1A2) gels, transferred onto nitrocellulose,
and probed with antibodies against rat Mrp2/cMoat (EAG15) and rat
CYP1A2 (MAb 1-7-1). Detection was performed using the ECL method, and
immunoreactive bands were analyzed by densitometry (mean ± S.D.).
*, PV absorbance is significantly different (p < .05, paired Student's t test) from that of PP.
|
|
| |
Discussion |
We had previously demonstrated utility of transport data
from isolated rat hepatocytes and in vitro data on the spontaneous and
enzymatic GSH conjugation in the prediction of GSH conjugation of EA in
the whole liver (Tirona and Pang, 1999). It was concluded that various
acinar factors on uptake, GSH availability, and distributions of the
GSTs and GSH conjugate efflux systems among zonal cells could affect
GSH conjugation. Although the identity of the EA transporter is
uncertain and may be similar to that for bumetanide (Horz et al.,
1996), a sodium-independent saturable system that is inhibitable by
organic anions was found to exist for EA uptake with rat hepatocytes
(Tirona and Pang, 1999). The EA transporter appears to be homogeneously
distributed within the liver acinus because there is no difference for
EA uptake among the enriched PP and PV hepatocytes (Fig. 2). The
kinetic parameters obtained from uptake experiments were similar among
the zonal regions and were not different than those obtained from
hepatocytes prepared from all zonal regions (Tirona and Pang, 1999).
Next to be considered is the aspect of zonal metabolism. In contrast to
our anticipation of observing differences in metabolism at 200 µM EA
(Tirona and Pang, 1999), initial rates of GSH conjugation within the PP
and PV hepatocyte incubation were similar and closely resembled those for transport (Table 2). Moreover, GSH depletion rates were not different among PP and PV hepatocytes treated with EA (Figs. 3A and
4A), suggesting the absence of zonal influence by GSH. The EA-SG
formation rates were, however, much lower than those found in the
corresponding cytosolic fractions of PP and PV hepatocytes (Table 2),
inferring strongly that hepatocyte EA uptake rate-limits GSH
conjugation regardless of the zonal position along the sinusoidal plate.
The difference in GST activities within the cytosolic fractions
of the PP and PV hepatocytes could have been revealed in the in vitro
studies, except for the shallow or modest gradient in PV enrichment of
GST activities toward EA and CDNB (Fig. 5). The results on GST
activities coincided with immunodetectable GST Ya and Yb2 levels
measured from cytosols of enriched PP and PV hepatocytes (Fig. 6). As
demonstrated by others for CDNB (Kera et al., 1987; Suolinna et al.,
1989), the PV enrichment of cytosolic GST activities was modest (1.2- to 1.6-fold). The PP and PV lysates obtained by the
dual-digitonin-pulse perfusion (Quistorff and Grunnet, 1987) offer an
improved method for the study of metabolic heterogeneity in liver. With
this technique, provision of the cellular contents of the most proximal
or distal hepatocytes is accomplished by controlling the depth of
digitonin penetration. These lysates proved to be more accurate in
relating to PP and PV activities and showed 2- to 3-fold PV
predominance in GST activity toward EA and CDNB (Fig. 5) and complement
corresponding changes in immunoreactive GSTs Ya and Yb2 in the lysate
(Fig. 7). These results, along with those from isolated PP and PV
hepatocytes, confirm that acinar gradients in GST activity toward EA
and CDNB exist, and the gradients are relatively shallow. Comparable
observations were reported for CDNB GST activities (Kera et al., 1987)
and GST protein contents in PP and PV lysates (Lindros et al., 1998). The slightly lower GST activity in PP lysate compared with PV lysate
(155 ± 44 and 364 ± 42 nmol/min/mg cytosolic protein
respectively, Fig. 6A) toward EA would not rate-limit EA elimination
because the transport activity is much lower.
Our metabolic data demonstrate that limitations existed in the
intact, isolated PP and PV hepatocyte system for the study of
functional metabolic heterogeneity. In examination of the PP marker
enzyme ALT, the PP/PV activity ratio (~1.8) normally obtained was
considerably lower than that (~7.5) in lysates (Table 1). This
difference underscores the fact that enriched zonal hepatocytes are
harvested from approximately half of the sinusoidal length and are
inevitably cross-contaminated to a higher degree (especially from
midzonal regions) than for lysates, which are derived from only a small
population of hepatocytes at the most distal and proximal acinar
regions. Therefore, depending on the shape and steepness of the zonal
distribution of enzymatic activities, difficulties persist in
identifying shallow gradients on metabolic activities, as found in this
study. However, the enrichment in the PV/PP ratio of GS was not much
improved with the lysate over the hepatocytes because the distribution
of GS is mostly confined to the last cells around the hepatic venules
(Burger et al., 1989) and will not be perturbed much by the
contamination of cells from other zonal regions in which GS was absent.
Analogously, we cannot exclude the possibility that a shallow gradient
exists for EA transporters, although we did not observe a difference in
EA uptake among the zonal cells. There also is the possibility that the
contents of nonparenchymal cells are sampled during
dual-digitonin-pulse perfusion; this may also contribute to the
differences seen in the metabolic activities observed in lysates and
hepatocyte cytosols.
The aspects of accumulation and efflux of EA-SG were further
addressed. Because product inhibition of the GSTs by EA-SG was known to
exist (Ploemen et al., 1990, 1993), Mrp2 may play a role in GSH
conjugation due to the removal of EA-SG at the canalicular membrane. We
have shown that 90% of the formed EA-SG was rapidly excreted into bile
of the rat liver preparation (Tirona and Pang, 1999), suggesting that
canalicular transport predominates over cellular efflux mediated
perhaps by the Mrp family proteins present on the lateral or
basolateral membranes (Zaman et al., 1997). Although human MRP2 was
found to transport EA-SG (Evers et al., 1998), rat Mrp isoforms have
not been directly shown to transport EA-SG. However, there appears to
be a close substrate specificity between the human and rat orthologs
(Keppler et al., 1998), and ATP-dependent uptake of the typical Mrp2
substrate, dinitrophenyl-GSH conjugate, into rat canalicular membrane
vesicles was almost completely inhibited by EA-SG (Ballatori and
Truong, 1995). It is hence reasonable to assume that Mrp2 is primarily
responsible for the efflux of EA-SG by hepatocytes. From our studies,
it was noted that the initial EA-SG efflux rates among PP and PV
hepatocytes were similar (Table 2), suggesting homogeneity in Mrp2
function within the liver acinus. The observation is consistent with
the lack of zonal differences in the levels of immunoreactive Mrp2
protein as determined by immunoblotting (Fig. 8) and acinar homogeneity
in MRP2 described by Kool et al. (1999) in human liver using an
immunohistochemical technique. Recently, Morrow et al. (1998)
demonstrated that the combined effect of enhanced expression of GST
P1-1 and MRP1 in MCF7 breast carcinoma cells conferred resistance to
the cytotoxic effects of EA. Elevated expression of either GST P1-1 or
MRP1 alone did not cause significant resistance to toxicity. Given the
dual presence of GSTs and Mrp2 in hepatocytes throughout the acinus,
all hepatocytes are amply endowed with cytoprotective defenses against
exposure to electrophiles such as EA.
The greater GSH conjugation over the efflux activity usually
suggests an accumulation of EA-SG; however, this was not observed. EA-SG accumulated only transiently at the onset of incubation within
isolated zonal hepatocytes due to the cessation of EA-SG formation as
GSH became depleted and due to the rapid efflux into extracellular
medium (Figs. 3 and 4). It remained plausible that product inhibition
of the GSTs ensued during the early time period. The cessation of EA-SG
formation and rapid efflux brought about the decline in intracellular
concentrations, but EA-SG persisted within the cell after 20 min,
albeit at a low and constant level. This observation is consistent with
findings on the redistribution or internalization of Mrp2 from the cell
surface to endosomes and lysosomes shortly after isolation of the
hepatocytes (Oude Elferink et al., 1993; Roelofsen et al., 1995),
although in the native intact liver, the majority of Mrp2 protein is
localized on the apical membrane. Therefore, the intracellular EA-SG
remaining beyond 20 min of incubation most likely signifies that the
conjugate is sequestered within the intracellular vesicles (Fig. 1).
Indeed, the fitting exercise supports the above conjecture. Absence of the sequestered pool or intracellular vesicles inevitably led to poorer
fits to the data.
In conclusion, we demonstrated that the GST activities responsible for
the GSH conjugation of EA were high, and that a relatively shallow and
increasing acinar gradient existed in rat liver. Despite this zonal
heterogeneity in metabolic activity, uptake was uniform throughout the
acinus, and this, together with low GSH levels, rate-limited the GSH
conjugation of EA. The major efflux system for EA-SG occurs most likely
via Mrp2, and acinar homogeneity in Mrp2 function was observed.
However, the present study dealt only with uptake and metabolism in
hepatocytes. In extrapolating data to the whole liver, one must be
cognizant of the impact of nonparenchymal cells (Kupffer, Ito, and
endothelial cells), which also contain GSTs, albeit at reduced levels
(Steinberg et al., 1989, Parola et al., 1993, Lee et al., 1994). The
presence of other cell types and associated heterogeneities might
further affect the hepatic GSH conjugation of EA.
We thank Dr. Dietrich Keppler (Deutsches Krebsforschungszentrum,
Heidelberg, Germany) and Dr. Harry V. Gelboin (National Cancer Institute, National Institutes of Health, Bethesda, MD) for providing us with the antibodies toward Mrp2/cMoat and CYP1A, respectively, and
Dr. J. H. T. M. Ploemen (TNO, Ziest, the Netherlands)
for supplying the [14C]EA.
GSH, reduced glutathione;
GSSG, oxidized
glutathione;
EA, ethacrynic acid;
EA-SG, ethacrynic acid-glutathione
conjugate;
CDNB, 1-chloro-2,4-dinitrobenzene;
PP, periportal;
PV, perivenous;
ALT, alanine aminotransferase;
GS, glutamine synthetase;
CYP, cytochrome P-450;
Mrp2, rat multidrug resistance protein 2;
MRP2, human multidrug resistance protein 2;
cMoat, rat canalicular
multispecific organic anion transporter;
GST, glutathione
S-transferase.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
![<FENCE>−<FR><NU>V<SUP><IT>metab</IT></SUP><SUB><UP>max</UP></SUB>[EA]<SUB>C</SUB>[GSH]<SUB>C</SUB></NU><DE>K<SUP>GSH</SUP><SUB>m</SUB>K<SUP>EA</SUP><SUB>m</SUB>+K<SUP>EA</SUP><SUB>m</SUB>[GSH]<SUB>C</SUB>+K<SUP>GSH</SUP><SUB>m</SUB>[EA]<SUB>C</SUB>+[EA]<SUB>C</SUB>[GSH]<SUB>C</SUB></DE></FR></FENCE>/V<SUB>C</SUB>](/content/vol291/issue3/fulltext/1210/img005.gif)
![+<FR><NU>V<SUP><IT>metab</IT></SUP><SUB><UP>max</UP></SUB>[EA]<SUB>C</SUB>[GSH]<SUB>C</SUB></NU><DE>K<SUP>GSH</SUP><SUB>m</SUB>K<SUP>EA</SUP><SUB>m</SUB>+K<SUP>EA</SUP><SUB>m</SUB>[GSH]<SUB>C</SUB>+K<SUP>GSH</SUP><SUB>m</SUB>[EA]<SUB>C</SUB>+[EA]<SUB>C</SUB>[GSH]<SUB>C</SUB></DE></FR>](/content/vol291/issue3/fulltext/1210/img007.gif)
![−CL<SUP>EA-SG</SUP><SUB><IT>efflux</IT></SUB>[EA-SG]<SUB>C</SUB>−CL<SUP>EA-SG</SUP><SUB><IT>ves</IT></SUB>[EA-SG]<SUB>C</SUB>)/V<SUB>C</SUB>](/content/vol291/issue3/fulltext/1210/img008.gif)
