Nifedipine modulation of biliary GSH and GSSG/ conjugate
efflux in normal and regenerating rat liver
Bo
Yang and
Ceredwyn E.
Hill
Gastrointestinal Diseases Research Unit and Department of
Physiology, Queen's University, Kingston, Ontario K7L 5G2, Canada
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ABSTRACT |
Canalicular glutathione
secretion provides the major driving force for bile acid-independent
bile flow (BAIF), although the pathways involved are not established.
The hypothesis that GSH efflux proceeds by a route functionally
distinct from the high-affinity, low-capacity, mrp2-mediated pathway
was tested by using perfused rat liver and three choleretic compounds
that modify biliary secretion of GSH (the dihydropyridine nifedipine
and organic anion probenecid) or GSSG [sodium nitroprusside (SNP)].
Whereas nifedipine (30 µM) stimulated GSH secretion and blocked
SNP-stimulated GSSG efflux and choleresis, SNP (1 mM) was ineffective
against nifedipine-stimulated GSH efflux or BAIF, suggesting that most
GSSG exits through a GSH-inhibitable path independent of high-affinity
GSSG/glutathione conjugate transport. Three observations support this
proposal. SNP, but not nifedipine, significantly inhibited
bromosulfophthalein (BSP, 1 µM) excretion. Probenecid (1 mM) blocked
resting or nifedipine-stimulated GSH secretion but only weakly
inhibited BSP excretion. Glutathione, but not BSP, efflux capacity was
reduced following partial hepatectomy. We suggest GSH efflux is
mediated by a high-capacity organic anion pathway capable of GSSG
transport when its high-affinity route is saturated.
probenecid; choleresis; regeneration; multidrug
resistance-associated protein 2/ATP-dependent multiorganic anion
transporter
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INTRODUCTION |
PRIMARY
CHOLERESIS, or stimulation of bile production at the level of the
hepatocyte, is caused by increased apical transport of bile acids and
other osmotically active compounds into the biliary lumen or
canaliculus (21). Glutathione secretion accounts for at
least 25% of basal bile acid-independent bile flow in the isolated
perfused rat liver (5, 19). GSSG and other glutathione conjugates are transported across the canalicular membrane by the
ATP-dependent multiorganic anion transporter (cMOAT) (24), recently cloned and identified as the multidrug resistance-associated protein MRP2 (25). At higher (>0.1 mM) concentrations,
GSSG/conjugates are transported by an ATP-independent process that is
inhibited by millimolar levels of GSSG, GSH, or organic anions
(19, 25).
Canalicular GSH transport is not as clearly defined as that for its
oxidized form, although it provides ~80% of the biliary glutathione
pool. A yeast homolog of MRP2 has been shown to transport GSH, leading
to the suggestion that the hepatic isoform may also be capable of such
activity (27). The evidence for this is persuasive, albeit
indirect. First, cells engineered to overexpress MRP2 also have much
greater GSH transport capability than native cells (26, 34); second, the natural mrp2 mutation in
TR
rats results in greatly diminished GSH secretion
(16, 25); third, endotoxin exposure reduces both
immunoreactive mrp2 in the canalicular membrane and total glutathione
secretory rate (33, 35); and fourth, chronic feeding of
mice with mrp2-inducing chemicals leads to increased biliary
secretion of GSH (37). Conversely, GSH transport in
canalicular membranes of normal and TR
rats is not
significantly different (7), and biliary secretion of GSH
is depressed in partially hepatectomized rats in the absence of changes
in mrp2 expression or bilirubin excretion (8, 15, 36). To
add to the complexity, there is some evidence that hepatic GSH is
transported by both high-affinity and low-affinity processes (3). Some reports suggest that both processes are mediated by mrp2 (26, 34). In any case, functional differentiation between GSSG/glutathione conjugate efflux and GSH secretion in the
normal rat liver has not been demonstrated.
Bile acid-independent choleresis is also induced by other compounds
synthesized by the liver such as nitric oxide (NO). The choleresis caused by exogenous, perfused NO donors is coupled to
enhanced GSSG but not GSH secretion (14, 32). In both
wild-type and TR
rats (that do not secrete GSSG in
response to NO), NO exposure roughly doubles the tissue levels of GSSG
without significantly affecting the total glutathione pool
(32). Thus high levels of NO appear to induce an oxidative
stress response, which may be buffered by mrp2-mediated excretion of
GSSG and subsequent stimulation of transport away from the hepatocyte.
The effect of acute exposure to NO donors on GSSG secretion and bile
acid-independent bile flow in the regenerating liver has not been reported.
Here we show that the two choleretic compounds nifedipine and
probenecid have opposite effects on biliary GSH secretion in the
perfused rat liver. We used these compounds, along with the NO donor
sodium nitroprusside (SNP), to investigate the processes responsible
for the secretion of GSH and GSSG/glutathione conjugates in the intact
perfused rat liver. The results suggest that GSH is transported by a
mechanism that can also move GSSG, the latter at lower relative
affinity and under conditions in which the high-affinity GSSG
transporter cMOAT/mrp2 is saturated. Thus the GSH pathway would provide
an overflow route for excess GSSG during conditions leading to
increased NO production (hepatic inflammation), and L-type
Ca2+ channel blockers such as nifedipine may attenuate
injury by maintaining intra- and/or extracellular GSH levels.
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MATERIALS AND METHODS |
Animals and materials.
Male Sprague-Dawley rats weighing 200-225 g (Charles River
Laboratories, Montreal, PQ) were maintained on a 12:12-h light/dark cycle with access to rat chow and water ad libitum according to the
regulations of the Animal Care Committee of Canada. Nifedipine was
purchased from Research Biochemicals, and glutathione reductase and
NADPH were from Roche Biochemicals (Montreal, PQ); all other chemicals
were from Sigma Chemical (St. Louis, MO) or British Drug Houses
(Toronto, ON).
Liver perfusions.
Livers were perfused via the portal vein with Krebs-Henseleit
bicarbonate-buffered (KH) saline using a non-recirculated,
flow-constant perfusion system as described previously
(13). KH saline was warmed to 37°C, saturated with
95%/5% (vol/vol) O2/CO2,
and perfused at 4-5 ml · min
1 · g
liver
1. Tissue viability was assessed throughout each
perfusion by monitoring portal pressure (2.53 ± 0.13 cmH2O/g liver; n = 40). Bile samples were
collected over consecutive 3- or 5-min intervals from a cannula placed
in the common bile duct, and bile volume was determined assuming a
density of 1 g/ml. Samples, stored on ice, were immediately assayed for
GSH and within 6 h for total glutathione. Nifedipine was dissolved
in DMSO and diluted (1:1,000) into the perfusate. The perfusate
concentrations of nifedipine and DMSO were 30 µM and 0.1% (vol/vol),
respectively. DMSO at this concentration had no effect on portal
pressure or bile flow. Probenecid was dissolved in 0.1 M Tris, pH 8.0, before being diluted (1:400) into the KH saline. Tris alone (0.25 mM)
had no effect on glutathione secretion, bile flow, or portal pressure.
Neither nifedipine, probenecid, nor SNP infusion had any effect on
portal pressure. Livers were perfused for at least 20 min before
introduction of test substances into the KH saline.
Partial hepatectomy and perfusion in situ.
Animals were anesthetized with an intraperitoneal injection of
ketamine/xylazine (90/10 mg/kg body wt) following administration of an
analgesic (buprenorphine, 0.03 mg/kg ip). Two-thirds partial hepatectomy was performed as described (11). Sham
operations involved all procedures, with the exception of
externalization and removal of the median and left lateral lobes.
Perfusions were carried out as described above, except that the
perfusate flow rate was reduced to account for the decreased liver
mass. Under these conditions, portal pressure was 5.88 ± 0.14 cmH2O/g liver (n = 11). From body and liver
weight data collected before these experiments we calculated perfusion
flow rates, and after each we determined that flow was 4.49 ± 0.18 ml · min
1 · g liver
1.
Glutathione assay.
GSH was measured in bile samples immediately following collection by
diluting (1:200) into ice-cold 0.1 mM 4,4'-dipyridyldisulfide (DPS) in
0.1 M sodium phosphate buffer (pH 7.4) and monitoring the absorbance at
324 nm. After correcting for absorbance due to other
non-sulfhydryl constituents in bile (parallel samples were
assayed in the absence of DPS) and comparing optical density against
cysteine standards, the rate of secretion of reduced thiol was
calculated. Total glutathione (GSH + GSSG) was routinely assayed in bile samples by using the enzyme recycling method and was expressed as GSH equivalents (9). To check the reliability of the
DPS assay, in selected experiments bile was deproteinized by collection into 6% sulfosalicylic acid (SSA) in the absence (GSH + GSSG) or
presence (GSSG) of 2-vinylpyridine followed by processing through the
glutathione reductase recycling assay (9). In the latter experiments, acivicin (20 µmol/kg body wt) was instilled in a retrograde fashion over 1 min into the common bile duct, which was
occluded for an additional minute before cannulation to inhibit ectopic
-glutamyl transpeptidase (33). In rats 24 h after
partial hepatectomy, the amount and volume of acivicin were reduced by two-thirds to reflect the decrease in liver mass. Tissue levels of
glutathione were determined from liver samples removed at the termination of perfusion. Samples were immediately frozen in liquid N2 and then homogenized in 5 volumes of 6% SSA,
neutralized, and assayed for GSH and total glutathione using the DPS
and reductase assays, respectively.
Bromosulfophthalein analysis.
Bile samples were diluted (1:200) in 0.1 N NaOH, and the absorbance at
580 nm was recorded and quantified against known standards.
Statistical analysis.
Data are presented as means ± SD of at least three perfusions for
each condition. Student's t-test was used to compare pairs of samples, and P values <0.05 were considered significant.
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RESULTS |
Differential modulation of canalicular GSH, GSSG, and
bromosulfophthalein efflux by nifedipine and NO suggests the presence
of a conjugate-independent GSH transport pathway.
Although it is already established that nifedipine is choleretic at
doses expected to be seen in the clinical setting (12, 28), the mechanism responsible for this choleresis is not known. Figure 1A shows that 30 µM
nifedipine caused a peak stimulation of bile flow of 1.6-fold, from
1.1 ± 0.1 µl · min
1 · g
1 under
basal conditions to 1.8 ± 0.3 µl · min
1 · g
1
(n = 4) in the isolated perfused rat liver. This
choleresis is paralleled by an increase in the total glutathione
secretory rate (Fig. 1A), from 5.1 ± 0.4 nmol · min
1 · g
1 under
basal conditions to 18.3 ± 3.4 nmol · min
1 · g liver
1 at
32.5 min of perfusion. The increase in total glutathione could be
accounted for by an increase in GSH secretion from 3.0 ± 1.1 to 15.8 ± 4.5 nmol · min
1 · g
1 (Fig.
1A), suggesting that a major fraction of the choleretic potential of nifedipine results from its stimulation of GSH secretion. Conversely, the NO donor SNP is choleretic as a result of its stimulation of GSSG excretion without affecting GSH efflux (Fig. 1B), as has already been reported for SNP and other NO
donors (14, 32). Furthermore, GSSG appeared in bile at
more than double the rate of nifedipine-induced GSH efflux. These
distinct effects of nifedipine and SNP on GSH and GSSG secretion were
exploited to determine whether these are functionally competitive
processes or whether they are mediated by independent pathways.

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Fig. 1.
Effects of nifedipine or sodium nitroprusside (SNP) on
bile flow and biliary glutathione secretion. Livers were perfused in
the absence (open symbols; n = 3) or presence (solid
symbols; n = 4) of either 30 µM nifedipine between 20 and 70 min (A) or 1 mM SNP between 35 and 55 min
(B). Mean (± SD) rates of bile flow, total glutathione
(GSH + GSSG), and reduced glutathione (GSH) efflux are plotted.
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In the majority of our experiments, we used DPS as a rapid method to
trap GSH. Additionally, we did not routinely inhibit canalicular
-glutamyl transpeptidase activity or acidify bile samples. To
determine whether our approach significantly affected the outcome of
the glutathione assays, we performed the experiments illustrated in
Fig. 1 following retrograde infusion of acivicin to inhibit ectopic
transpeptidase and collection of bile into SSA to inhibit GSH
oxidation. Figure 2 shows the basal,
SNP-, and nifedipine-stimulated bile flow rates and secretion of total glutathione and GSH following acivicin/acidification exposure compared
with non-pretreated samples. Mean basal rates of total glutathione and
GSH secretion but not bile flow were significantly higher in the
pretreated liver and bile samples. In contrast, nifedipine- or
SNP-stimulated bile flow and, respectively, GSH or total glutathione
secretion were not significantly affected by the retrograde infusion of
acivicin and subsequent acidification. These results confirm an earlier
report showing that acivicin had no significant effect on SNP-induced
glutathione secretion in bile samples collected into acid
(33). Therefore, for the remainder of the experiments in
which the normal rat liver was used, the acivicin infusion and bile
acidification steps were omitted, and the DPS assay was used for rapid
monitoring of GSH secretion.

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Fig. 2.
Effect of acivicin/acidification pretreatment on biliary
glutathione fractions in nifedipine- and SNP-perfused rat liver. Livers
were perfused in the absence of acivicin (open bars) or following
retrograde infusion of acivicin (20 µmol/kg) (hatched bars) with
either 1 mM SNP (A) or 30 µM nifedipine (NIF;
B) between 35 and 55 min. Bile samples were collected into
ice-cold 4,4'-dipyridyldisulfide (DPS; acivicin) or sulfosalicylic
acid (SSA; + acivicin) and assayed for total (SNP-treated livers) or
reduced (NIF-treated livers) glutathione. Mean (± SD;
n = 4 for each condition) rates of bile flow and total
(GSSG + GSH) or reduced (GSH) glutathione secretion under basal
(at 32.5 min of perfusion) or stimulated (52.5 min) conditions were
plotted.
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To determine whether GSH efflux is mediated by a GSSG transporter,
experiments were designed to identify the existence of mutual
competition between the efflux of SNP- and nifedipine-stimulated GSSG
and GSH, respectively. When SNP was infused after the secretory response to nifedipine had reached a steady state, a small increase in
mean choleresis was observed, although this was not significant at any
time point (Fig. 3). No significant
change in GSH or additional GSSG excretion was observed, indicating
that SNP-induced GSSG release is completely inhibited by nifedipine,
whereas GSH efflux induced by the latter is not significantly affected
by SNP.

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Fig. 3.
Nifedipine inhibits SNP-induced biliary GSSG efflux and
choleresis. Livers were perfused in the presence of 30 µM nifedipine
between 20 and 70 min, either in the absence (open symbols, same data
as Fig 1A solid symbols) or presence (solid symbols;
n = 4) of 1 mM SNP between 35 and 55 min. Mean (± SD)
rates of bile flow, total (GSSG + GSH), and reduced (GSH)
glutathione efflux were plotted.
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To determine whether nifedipine- or SNP-stimulated GSH or GSSG
secretion is mediated by the high-affinity, low-capacity
cMOAT/mrp2 route, bromosulfophthalein (BSP) excretion was
monitored in the absence or presence of nifedipine or SNP. BSP was
infused at 1 µM, which is 30-fold less than the apparent
Michaelis-Menten constant (Km) for
high-affinity, ATP-dependent efflux of this compound in
isolated canalicular membranes (22). BSP efflux was
significantly inhibited by SNP but not by nifedipine (Fig.
4A). As summarized in Table
1, SNP blocked 55.3 ± 9.8% of BSP
excretion, whereas nifedipine did not significantly alter BSP efflux.
Furthermore, nifedipine pretreatment did not affect the SNP inhibition
of BSP excretion (Fig. 4B; see Table 1). These results
suggest that GSH secretion stimulated by nifedipine is not mediated by
mrp2, whereas SNP and BSP generate glutathione conjugates that are
transported through mrp2.

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Fig. 4.
Effects of nifedipine and SNP on biliary excretion of
tracer amounts of bromosulfophthalein (BSP). Livers were perfused with
1 µM BSP between 20 and 83 min (A) or 20 and 92 min
(B), and bile samples were analyzed for BSP/BSP conjugate.
In A, livers were perfused in the absence ( ;
n = 3) or presence of 30 µM
nifedipine ( ; n = 3) or 1 mM SNP
( ; n = 3) between 41 and 62 min. In
B, livers were perfused with 30 µM nifedipine between 41 and 92 min and 1 mM SNP between 56 and 77 min (n = 3).
Mean (± SD) rates of BSP efflux were plotted.
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Probenecid block of basal and nifedipine-stimulated GSH secretion
but not BSP excretion suggests independent efflux pathways for GSH and
BSP.
The organic anion probenecid is excreted into bile as the unchanged
anion and its glucuronic acid conjugate in a 1:2 ratio (10). Figure 5 shows that 1 mM probenecid is choleretic without inhibiting tracer (1 µM) BSP
excretion. The amount of BSP excreted between 41 and 62 min changed
from 3.3 ± 0.3 nmol · g
1 · min
1
(n = 3) under basal conditions (Table 1) to 2.9 ± 0.2 nmol · g
1 · min
1
(n = 3) in the presence of probenecid. This minimal
effect on BSP excretion (as was also seen in response to nifedipine)
suggested that probenecid efflux occurs by a pathway separate from the
high-affinity mrp2 route and therefore might competitively inhibit
nifedipine-induced GSH secretion. Furthermore, if GSH and organic
anions are transported by the same organic anion transporter,
probenecid should inhibit basal GSH secretion.

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Fig. 5.
Effects of probenecid on bile flow and BSP excretion.
Livers were perfused in the presence of 1 µM BSP between 20 and 83 min and 1 mM probenecid between 41 and 62 min (n = 3).
Mean (± SD) rates of bile flow (top) and BSP/BSP conjugate
efflux (bottom) were plotted.
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When 1 mM probenecid was perfused between 35 and 85 min (Fig.
6A) it caused a reversible
choleresis that was accompanied by a 76.0% decrease in GSH release,
suggesting that excretion of this organic anion directly competes with
GSH efflux. The choleresis was not due to enhanced GSSG efflux (not
shown), and therefore it was likely a result of the excretion and
biliary accumulation of probenecid and its glucuronic acid conjugate.
Further support for a common GSH/probenecid transporter was gained from
experiments in which probenecid and nifedipine (30 µM) were
alternately perfused and choleresis and GSH secretion were measured
(Fig. 6). Whether probenecid was perfused first or during steady-state
stimulation of GSH secretion by nifedipine, the nifedipine-induced GSH
efflux was inhibited by 75.7%, suggesting competitive interaction at the level of the GSH transport pathway.

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Fig. 6.
Effects of probenecid and nifedipine on bile flow and GSH
secretion. A: livers were perfused in the presence of 1 mM
probenecid between 35 and 85 min in the absence (open symbols;
n = 3) or presence (solid symbols; n = 3) of 30 µM nifedipine between 50 and 70 min. B: livers
were perfused in the presence of 30 µM nifedipine between 35 and 85 min with 1 mM probenecid (n = 5) between 50 and 70 min.
Mean (± SD) rates of bile flow and GSH efflux were plotted.
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Canalicular glutathione transport modulated by nifedipine,
probenecid, and SNP is not limited by changes in steady-state levels of
tissue glutathione.
NO increases GSSG efflux in the isolated, perfused rat liver as a
consequence of oxidative stress-induced accumulation of GSSG (see Ref.
32 and references therein). Table
2 shows that 15 min after cessation of
SNP perfusion, tissue glutathione levels are not significantly
different from control tissue. To determine whether nifedipine in the
absence or presence of SNP or probenecid affected GSH transport
activity as a result of perturbation of cellular glutathione pools,
tissue levels of GSH and total glutathione were measured at the end of
each group of perfusions. Table 2 shows that total glutathione,
determined by an enzymatic assay, and GSH, determined by an
acid-soluble thiol trapping technique, were not significantly different
under any of the conditions used in this study. These results indicate
that the acute increase in GSH or GSSG efflux rate in response to
nifedipine or SNP, respectively, reflects small changes in flux through
the glutathione pool that are compensated for by a mass action response
at the transporter level (i.e., efflux is not rate limiting).
Consequently, the cellular GSH/GSSG steady state is maintained. The
lack of a significant increase in tissue GSH consequent to inhibition
of canalicular efflux by probenecid suggests, again, that flux through
the tissue glutathione pool changes to maintain a steady state.
Short-term regeneration is associated with decreased glutathione
secretion but not BSP efflux.
One day after partial hepatectomy, the regenerating liver is
characterized by a twofold increase in tissue GSH and a 50% reduction in canalicular GSH secretion in the absence of significant changes in
mrp2 expression or activity (15, 35), indicating that the canalicular GSH efflux pathway is depressed preferentially over mrp2.
We extended these observations to the sham-operated or partially hepatectomized liver perfused in the absence of bile salts (i.e., depressed bile flow was not compensated for by bile acid perfusion or
an intact circulation). Figure 7
demonstrates that BSP excretion in the regenerating rat liver is not
significantly different between sham-operated and partially
hepatectomized animals. These results extend the earlier reports by
specifically demonstrating that high-affinity, low-capacity (mrp2)
activity is not perturbed following partial hepatectomy.

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Fig. 7.
Effect of partial hepatectomy (PH) on BSP excretion and
bile flow. Livers 24 h after PH (solid symbols; n = 3) or sham operation (open symbols; n = 3) were
perfused in the presence of 1 µM BSP between 20 and 59 min. Mean (± SD) rates of bile flow (top) and BSP/BSP conjugate efflux
(bottom) were plotted.
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Our results also confirm earlier reports (15) that
regeneration is accompanied by decreased GSH secretion and bile flow (Fig. 8, 20- to 35-min intervals). As
summarized in Table 3, mean basal bile
production, GSH, and total glutathione secretion were decreased
significantly by 24, 43, and 57%, respectively, in regenerating livers
compared with sham-operated animals. The effect of nifedipine or SNP on
bile flow and glutathione secretion was also monitored in regenerating
livers. The time courses are illustrated in Fig. 8, and the mean rates
of GSSG or GSH released per gram liver in the presence of nifedipine or
SNP are summarized in Table 3. Mean choleresis induced by nifedipine or
SNP was between 30 and 35% lower in the regenerating livers compared
with the sham-operated animals. GSH and total glutathione were
decreased by 45 and 52% in response to nifedipine and SNP,
respectively. However, although the rates of glutathione are decreased
~50% in the regenerating livers, the percent stimulation by
nifedipine or SNP is not significantly different between these and
livers of sham-operated animals (Table 3). This suggests that the
functional transporters in the remnant liver, although reduced in
number by roughly half, are capable of activity similar to those in
sham-operated controls.

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Fig. 8.
Effect of PH on nifedipine- or SNP-stimulated glutathione
secretion and bile flow. Livers 24 h after PH (solid symbols;
n = 4/group) or sham operation (open symbols;
n = 4/group) were perfused in the presence of 30 µM
nifedipine (A) or 1 mM SNP (B) between 35 and 55 min. Mean (± SD) rates of bile flow (top) and efflux of GSH
(A, bottom) or GSH + GSSG (B,
bottom) were plotted.
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Lastly, we assessed the potential effect of partial hepatectomy on
biliary glutathione stability, since canalicular
-glutamyl transpeptidase activity has been shown to increase in other models of
impaired bile acid-independent flow (33). Figure
9 shows the rates of SNP-stimulated bile
flow and total glutathione efflux in livers from regenerating vs.
sham-operated animals. Livers were perfused and bile samples were
collected in the absence or presence of acivicin and SSA. The latter
treatment decreased bile flow under basal and SNP-stimulated conditions
in the partially hepatectomized livers but did not significantly affect
the sham-operated animals. Similar trends were observed for total
glutathione secretion, suggesting that retrograde infusion of acivicin
into the bile duct of the regenerating liver compromises bile flow and
inhibits glutathione secretion, possibly as a result of backflow
through the weakened paracellular junctions of the regenerating liver. We conclude that the results obtained in the absence of
acivicin/acidification are likely more representative of bile
acid-independent bile flow in the isolated, perfused rat liver 24 h after partial hepatectomy.

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Fig. 9.
Effect of acivicin/acidification pretreatment on
basal and SNP-stimulated total biliary glutathione secretion in
sham-operated and PH rat liver. Livers were perfused in the absence of
acivicin (open bars) or following retrograde infusion of acivicin (20 µmol/kg) (hatched bars) (n = 6 for each group) with 1 mM SNP between 35 and 55 min. Bile samples were collected between 20 and 35 min (Basal), 35 and 55 min (+SNP), and 55 and 70 min (Recovery)
into DPS ( acivicin) or SSA (+acivicin) before assay for total
glutathione. Mean (± SD) rates of bile flow and GSH + GSSG efflux
were plotted.
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DISCUSSION |
In this study, we demonstrate that the L-type calcium channel
blocker nifedipine and the anion transport inhibitor probenicid are
choleretic in spite of their abilities, respectively, to stimulate or
block the secretion of reduced glutathione into the biliary lumen. We
used these compounds, combined with a concentration of SNP already
established to stimulate near-maximum canalicular GSSG secretion
(32) and tracer amounts of a glutathione
conjugate-generating substance (BSP), to study the functional
relationship between GSSG/conjugate and GSH transport in the intact,
nominally bile acid-free, perfused liver. This was pursued because
although GSH secretion provides a significant driving force for
canalicular bile acid-independent bile formation (5), the
molecular processes are not yet understood, and the physiological
significance of those that have been reported from studies in vitro is
not established.
Our results support the following functional model of glutathione
secretion. This model predicts two major routes for GSH, GSSG, and
glutathione conjugate efflux across the canalicular membrane. Under
basal conditions, GSSG/conjugates are transported by a high-affinity
but low-capacity route that is GSH insensitive and mediated by mrp2
(17). Under conditions in which cytosolic GSSG
concentration is increased (e.g., oxidative stress and increased NO
generation), the low-capacity route becomes saturated and excess GSSG
overflows through the GSH transporter, operationally defined as cgsht
(see Ref. 4). This route also serves as the major conduit
for GSH efflux. It has a relatively high capacity and higher affinity
for GSH and unconjugated organic anions than oxidized glutathione and
glutathione conjugates. This model, and the results presented herein,
provide functional evidence supporting some of the pathways described
in canalicular membrane vesicles and demonstrate that specific drugs
can independently modulate these routes. The model will be discussed in
terms of the existing studies in vitro and the data reported here.
The model proposes that the major route for GSH efflux is a
high-capacity path also capable of transporting organic anions and, at
lower affinity, glutathione disulfide and glutathione conjugates.
Studies using canalicular membranes reported that GSH secretion occurs
via ATP-independent and potential-sensitive high
(Km ~ 0.25mM) and low
(Km ~ 17mM) affinity components, the latter having 25 times the capacity of the former (3, 7). The high-affinity path is inhibited by glutathione conjugates (3), whereas that for low affinity secretion is sensitive
to millimolar concentrations of organic anions (3, 7).
However, the contribution of the high-affinity route to GSH secretion
in the intact liver is minor since organic anions, but not their conjugates or GSSG, inhibit biliary GSH secretion in the perfused liver
(2, 18). Similarly, we found that GSH efflux under basal
conditions or in the presence of nifedipine was inhibited by the
organic anion probenecid but not significantly affected by the GSSG
secretagog SNP. Probenecid was choleretic, as reported earlier, as a
result of its concentrative efflux into the biliary lumen as the
glucuronic acid conjugate and the unchanged organic anion in a roughly
2:1 ratio (10). Thus organic anion/glucuronyl conjugate
excretion, as represented by probenecid, directly competes with GSH
efflux. Our results also suggest that probenecid is not a strong
inhibitor of cMOAT/mrp2.
Two different models of canalicular GSH efflux have been proposed to
account for the high and low affinity processes demonstrated in
canalicular membranes. One, based on radiation inactivation studies,
suggests that two different transport proteins are involved (20), whereas the alternative, based on correlation of GSH
transport and mrp2 expression, suggests that mrp2 alone can account for both pathways (26). Support for the latter comes from the
report that mrp2 overexpression results in increased ATP-independent, low affinity GSH flux (34), thus potentially accounting
for the energy-independent transport seen in canalicular membranes. However, these data also show that mrp2-mediated GSH flux is
significantly cis- and trans-inhibited by,
respectively, physiological levels of glutathione conjugates
(micromolar) and GSH (millimolar) (34). Therefore, mrp2
would account for only a small fraction of GSH efflux in vivo. In view
of this data, the increased rates of GSH efflux in mrp2 overexpression
models, or lack of release in mrp2-deficient livers of TR
rats, cannot be adequately explained as resulting from mrp2-mediated high capacity GSH efflux. It would therefore be of interest to extend
the observations presented here and to determine whether nifedipine can
stimulate GSH efflux in mrp2-deficient rats.
The correlation between mrp2 expression and GSH transport may yet be
explained by a functional relationship between GSH and GSSG release
mechanisms such that the glutathione redox balance is maintained. This
is supported by the observation that although the total glutathione
level is doubled in TR
rat liver compared with controls,
the fraction of GSSG is not significantly different (32).
Thus, without a functioning transporter for the release of GSSG, an
intact GSH transporter, if present, would be inactive. In this case,
nifedipine would not be expected to significantly stimulate GSH efflux
in mrp2-deficient animals. The role of mrp2 in GSH efflux will be
equivocal until purified, reconstituted preparations of this
transporter are analyzed.
The model also predicts that GSSG or glutathione conjugates at high
relative concentrations are released through the high-capacity GSH
pathway. In favor of this is the block of near-maximal SNP-stimulated glutathione efflux by nifedipine (Fig. 3) and the minimal effect of
nifedipine on efflux of tracer amounts of BSP (Fig. 4), which is mainly
secreted as its glutathione conjugate through mrp2 (23, 30). The present results extend observations in canalicular vesicles showing that both GSH (1 mM) and organic anions
cis-inhibited the low-affinity, ATP-independent uptake of
both glutathione conjugates and GSH (3, 6).
Lastly, the model predicts that, under basal conditions, GSSG is
transported mainly by a GSH-insensitive, low-capacity route mediated by
mrp2. Since GSSG is transported against its concentration gradient into
the canaliculus, efflux must be an energy-requiring process.
ATP-dependent conjugate or GSSG transport in canalicular membranes
occurs through a high-affinity, GSH-insensitive process (1, 3, 22), with properties similar to mrp2-mediated flux (17). We monitored the activity of this transporter by
following the excretion of tracer concentrations of the glutathione
conjugate of BSP (BSP-SG). Whereas GSSG (from SNP) blocked the
major fraction of BSP excretion (Fig. 4), GSH (via nifedipine) and
organic anions (probenecid) were much less effective in inhibiting BSP
appearance in bile (Figs. 4 and 5). Since ~26% of nifedipine is
excreted into the bile as its glucuronyl conjugate and the remainder
mainly as carboxylate metabolites (29), the minor
inhibition of BSP excretion by nifedipine and probenecid could result
from competition with excretion of their conjugates through mrp2.
As an independent test of the model of GSH and GSSG/conjugate secretory
pathways, we followed BSP and glutathione efflux in the regenerating
liver perfused in the absence of added bile salts. Bile
acid-independent bile flow, extrapolated from flow vs. bile salt
secretion curves (38), and GSH secretion (15)
are decreased in the early stages of regeneration as confirmed here,
even though tissue glutathione is increased (15, 31).
Furthermore, mrp2 activity and expression, as assessed by Northern and
Western blot and functional studies (bilirubin excretion), are not
significantly affected by partial hepatectomy (8, 35).
Thus the low-affinity, high-capacity glutathione transport process,
rather than the high-affinity, low-capacity, ATP-dependent path,
appears to be selectively depressed during this period. Our model
predicts, then, that tracer BSP excretion should not be affected,
whereas GSH and GSSG, secreted by nifedipine and saturating amounts of
SNP, respectively, should be depressed. In support of this, both basal
and nifedipine-induced GSH secretion as well as SNP-augmented GSSG
efflux were all decreased at 24 h after partial hepatectomy (Fig.
8; Table 3), whereas tracer BSP excretion was not significantly
different between sham-operated and hepatectomized animals (Fig. 7).
Conversely, the percent stimulation of efflux above basal levels by
nifedipine or SNP was not significantly different between sham-operated
and regenerating livers (Table 3). These results suggest that, although
the residual low affinity, high capacity transporters are decreased in
number (as opposed to the BSP transporters), their functional activity
is not modified. In combination, these results provide further evidence
for the existence of a glutathione transporter distinct from mrp2.
Additionally, the GSH transporter appears to be more sensitive to
conditions in which polarized functions are compromised, such as regeneration.
In summary, we have identified a new GSH secretagog and used this to
define the major physiological transport processes taken by GSH and
GSSG/glutathione conjugates in their transfer into the biliary
compartment. These results support the proposal that two distinct
processes are responsible for GSSG/conjugate and GSH/organic anion
secretion. In addition, we have provided evidence for a
reinterpretation of the mechanism responsible for the cytoprotective effects of calcium channel blockers.
 |
ACKNOWLEDGEMENTS |
This research was supported by the Medical and Natural Sciences and
Engineering Research Councils of Canada. Thanks to the Jeanne Mance
Foundation, Hotel Dieu Hospital, for salary support.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: C. E. Hill, GI Diseases Research Unit, Hotel Dieu Hospital, 166 Brock St.,
Kingston, ON K7L 5G2, Canada (E-mail
hillc{at}post.queensu.ca).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 August 2000; accepted in final form 1 February 2001.
 |
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