From the Department of Medicine II-Grosshadern,
Klinikum of the University of Munich, 81377 Munich, Germany and the
Liver Center, Yale University School of Medicine,
New Haven, Connecticut 06510
Received for publication, September 26, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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Taurolithocholic acid (TLCA) is a potent
cholestatic agent. Our recent work suggested that TLCA impairs
hepatobiliary exocytosis, insertion of transport proteins into apical
hepatocyte membranes, and bile flow by protein kinase C The hydrophobic bile acid taurolithocholic acid
(TLCA)1 was identified as
a potent cholestatic agent 35 years ago (1, 2). However, the mechanisms
of this cholestatic effect are not yet clear (3, 4). TLCA induces
cholestasis at low micromolar concentrations in vivo (1) as
well as in the isolated perfused liver (5, 6) and in isolated
hepatocyte couplets (7) in vitro. TLCA impairs hepatobiliary
exocytosis, a key step for the insertion of apical carrier proteins
into their target membrane, and lowers the density of the apical
conjugate export pump, Mrp2, in canalicular membranes of liver cells in
association with reduced canalicular excretion of organic anions (6).
In parallel, TLCA modulates a number of signaling events in liver cells
that may contribute to membrane vesicle fusion and membrane protein
insertion; TLCA may (i) impair Ca2+ influx across
hepatocellular membranes (8-10), (ii) reduce hepatocellular membrane
binding of the Ca2+-sensitive Products of phosphatidylinositol-3 kinases (PI3Ks) are mediators of
diverse cellular functions and may also modulate secretory activity of
epithelial cells (12, 13). In hepatocytes, PI3K is involved in
taurocholic acid (TCA)-induced biliary bile acid secretion (14, 15).
Interestingly, products of PI3K, phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate, are potent stimuli of the
In contrast to TLCA, the hydrophilic bile acid ursodeoxycholic acid
(UDCA) is a potent anticholestatic agent and is used for the treatment
of a number of cholestatic disorders (21, 22). The taurine conjugate of
UDCA (TUDCA) reverses TLCA-induced cholestasis by
PKC In the present study we investigated the role of PI3K and PKC Materials--
Anti-PI3K p85, p110 Animals--
Male Sprague-Dawley rats (229 ± 16 g)
were obtained from Charles River (Sulzfeld, Germany). They were
subjected to a 12-h day-night rhythm with free access to rodent food
and water.
Isolated Rat Liver Perfusion--
The technical procedure used
has been described previously (6). Rats were anesthetized with sodium
pentobarbital (50 mg/kg of body weight, intraperitoneal). After
cannulation of the bile duct, the portal vein, and the inferior vena
cava, the latter was ligated above the right renal vein. The liver was
perfused with Krebs-Ringer bicarbonate solution (6) at 37 °C at a
constant flow rate of 4.0-4.5 ml/min/g of liver. Temperature and
perfusion pressure were continuously monitored and did not
significantly change during any of the experimental conditions chosen
in this study. Bile flow was measured gravimetrically in pretared
tubes. Hepatovenous efflux of lactate dehydrogenase was measured as an indicator of liver cell damage by use of a standard enzymatic test
(24). Two perfusion protocols were applied for determination of
(i) hepatobiliary exocytosis and (ii) secretion of the model Mrp2
substrate, 2,4-dinitrophenyl-S-glutathione (GS-DNP).
Protocol I; Hepatobiliary Exocytosis (6, 25)--
Livers were
preloaded with 5 mg/dl HRP, 1 g/dl bovine serum albumin for 25 min in a
recirculating Krebs-Ringer bicarbonate perfusion solution (40 ml/min).
The perfusion was then switched to a non-recirculating HRP- and bovine
serum albumin-free Krebs-Ringer bicarbonate perfusion, residual HRP in
the vascular bed was washed out for 5 min, and the PI3K inhibitor,
wortmannin (or the carrier Me2SO only, 0.001%, v/v), was
continuously infused to reach a final concentration of 100 nmol/liter
in the portal vein. After 5 min, bile acids (or the carrier
Me2SO only; 0.1%, v/v) were infused for 50 min at a
continuous rate into the perfusion medium to reach a final
concentration of 10 or 25 µmol/liter, respectively, in the portal
vein. At the end of the experiments, the left anterior liver lobe was
clamped and excised. A sample of about 200 mg wet weight was
immediately transferred into ice-cold homogenization buffer and was
homogenized for determination of PKC Protocol II; Organic Anion Secretion (6)--
Livers were
perfused in a non-recirculating fashion with HRP- and bovine serum
albumin-free Krebs-Ringer bicarbonate for 95 min. After 15 min, the
PI3K inhibitor, wortmannin, (or the carrier Me2SO only,
0.001%, v/v) was continuously infused for 80 min to reach a final
concentration of 100 nmol/liter in the portal vein. After 20 min, the
bile acids TLCA and TUDCA (or the carrier Me2SO only;
0.1%, v/v) were infused for 75 min at a continuous rate into the
perfusion medium to reach a final concentration of 10 or 25 µmol/liter, respectively, in the portal vein. After 45 min, CDNB, the
precursor of GS-DNP, was infused for 10 min to reach a final
concentration of 30 µmol/liter in the portal vein, at which
saturation of biliary GS-DNP secretion was observed in the perfused rat
liver (26). At the end of the experiments, the left anterior liver lobe
was clamped and excised. A sample of this lobe was immediately
shock-frozen in liquid nitrogen and stored at
Biliary HRP activity was determined spectrophotometrically using
4-aminoantipyrine as substrate and recording the linear change in
absorption at 510 nm for 3 min at a constant temperature of 25 °C
(6, 25). HRP (ng of protein/min/g of liver) was quantitated after
establishing HRP standard curves. Biliary HRP secretion was expressed
as % of secretion at min 45 (after HRP loading and washout period) to
correct for differences in base-line total HRP activity.
Biliary secretion of GS-DNP was determined spectrophotometrically (6,
26). 5 µl of bile were added to 1000 µl of H2O in a
cuvette. Absorption was measured at 335 nm, and biliary GS-DNP levels
(nmol/liter) were calculated using the formula c = E335Vtotal/
PI3K activity in hepatic tissue was determined by use of a PI3K assay.
In brief, 100 mg of shock-frozen tissue were homogenized in ice-cold
lysis buffer (1 ml/50 mg of tissue; HEPES, 50 mmol/liter; NaCl, 137 mmol/liter; CaCl2, 1 mmol/liter; MgCl2, 1 mmol/liter; Na4P2O7, 10 mmol/liter;
NaF, 10 mmol/liter; EDTA, 2 mmol/liter; tergitol Nonidet P-40, 1%
(v/v); glycerol, 10%, v/v; antipain, 10 mg/liter; aprotinin, 2 mg/liter; benzamidine, 9.6 mmol/liter; leupeptin, 10 µmol/liter;
Na3VO4, 2 mmol/liter; pepstatin, 0.73 µmol/liter; phenylmethylsulfonyl fluoride, 1 mmol/liter, pH 7.5). Samples were kept on ice for 30 min and were then centrifuged for 30 min (3.500 × g, 4 °C). For immunoprecipitation,
aliquots from the supernatant containing 500 µg of protein were
incubated with 4 µg of anti-PI3K p85, p110
PKB/Akt activity in hepatic tissue was determined by an immunoblotting
technique (27, 28). In brief, shock-frozen tissue (
Distribution of the protein kinase C isoform
PDK-I activity in hepatic tissue was determined by use of a PDK-1
immunoprecipitation kinase assay kit (Upstate, Lake Placid, NY). In
brief, shock-frozen liver tissue (100 mg/ml) was homogenized on ice in
buffer A (Tris, 50 mmol/liter; Triton X-100, 0.1%; EDTA, 1 mmol/liter;
EGTA, 1 mmol/liter; NaF, 50 mmol/liter; sodium
Isolation and culture of rat hepatocyte couplets was performed as
previously described (30). Cells were plated in 100-mm Petri dishes
(~10 × 104 cells/cm2) containing glass
coverslips and incubated at 37 °C in L-15 medium for
4 h in an air atmosphere.
Bile acid secretion by IRHC was assessed by measuring the
hepatocellular uptake and secretion of 1 µmol/liter cholylglycylamido fluorescein (CGamF) into the canalicular space as previously described (30). CGamF was synthesized according to Schteingart et al. (31) and was kindly provided by Dr. Alan Hofmann. Four hours after isolation, hepatocytes (on coverslips) were briefly transferred to HEPES buffer (30). Then, cells were pretreated for 15 min at
37 °C with (i) Me2SO (0.1%, v/v), (ii) 100 nmol/liter
wortmannin and Me2SO, (iii) Me2SO for 5 min,
and 2.5 µM TLCA (in Me2SO, 0.1%, v/v) for 10 min, (iv) 100 nmol/liter wortmannin and Me2SO for 5 min,
and 100 nmol/liter wortmannin and 2.5 µM TLCA (in
Me2SO, 0.1%, v/v) for 10 min, (v) Me2SO for 5 min and 5 µmol/liter TLCA (in Me2SO, 0.1%, v/v) for 10 min, and (vi) 100 nmol/liter wortmannin and Me2SO for 5 min, and 100 nmol/liter wortmannin and 5 µmol/liter TLCA for 10 min.
Cells were then transferred for 5 min to HEPES buffer containing 1 µmol/liter fluorescent CGamF at 37 °C to allow adequate loading of
the fluorescent bile acid and transferred back for 10 min to their
previous dishes (i-vi). Hepatocyte secretion was stopped by placing
coverslips in ice-cold HEPES buffer on ice, and cells were viewed
immediately on a Zeiss LSM 510 microscope (Thornwood, NY). Laser
settings were optimized for a dynamic range to avoid saturation of the
fluorescence. The same settings were used for all conditions. Cells
were analyzed on the confocal laser scanning microscope by one
investigator (C. J. Soroka) who was blinded to the
experimental conditions. Couplets were selected based upon the presence
of a well defined canalicular space as determined under bright field
optics. Images were then acquired with rapid scanning to avoid
quenching of the fluorescence. Quantitation of uptake (uptake = (F° cell + F° can)/µm2) and secretion (% secretion = [F° can/(F° cell + F° can)] × 100) of CGamF
was performed as previously published (30), except that NIH Image
software was used.
PKB/Akt activity in isolated rat hepatocytes was determined by an
immunoblotting technique (27, 28). In brief, 4 h after plating
(see above) cells were incubated for 5, 15, 30, and 60 min with
TLCA (5 µmol/liter; at concentrations >5 µmol/liter, TLCA caused
visible damage in isolated hepatocytes in short term culture), TUDCA
(10 µmol/liter), TCA (10 µmol/liter), or the carrier Me2SO only (control, 0.1%, v/v). Culture dishes were then
placed on ice, and cells were scraped and immediately shock-frozen
( Statistics--
Data are expressed as the mean ± S.D.
Results were compared between groups using an unpaired two-tailed
Student's t test or ANOVA when indicated. p < 0.05 was considered statistically significant.
Hepatobiliary Exocytosis in Perfused Rat Livers (Protocol
I)--
Bile flow was 1.0 ± 0.1 µl/min/g of liver
(n = 47) after loading of livers with HRP for 25 min
and a wash-out period of 5 min and remained stable for the following 50 min in control experiments (52.2 ± 4.4 µl/50 min/g of liver,
n = 5) in the presence of dimethyl sulfoxide
(Me2SO, 0.1%, v/v) (Fig.
1A). Wortmannin (100 nmol/liter) did not affect bile flow under basal conditions (54.9 ± 5.1 µl/50min/g of liver, n = 5; Fig.
1A). TLCA (10 µmol/liter) caused a decrease of bile flow
to 18% of controls (9.5 ± 1.6 µl/50 min/g of liver, n = 6, p < 0.01; Fig. 1A).
The TLCA-induced decrease of bile flow was partially reversed by
wortmannin; bile flow was 50% (26.2 ± 3.0 µl/50 min/g of
liver, n = 6, p < 0.01 versus TLCA) and 47% (24.6 ± 3.5 µl/50 min/g of
liver, n = 5, p < 0.01 versus TLCA) of controls when wortmannin was administered at
100 nmol/liter and 500 nmol/liter, respectively (Fig. 1A).
TUDCA (25 µmol/liter) increased bile flow to 139% that of controls
(72.7 ± 16.1 µl/50 min/g of liver, n = 5, p < 0.05, Fig.
2A). Wortmannin did not affect
TUDCA-induced bile flow (73.7 ± 14.4 µl/50 min/g of liver, n = 5; Fig. 2A). TUDCA reversed TLCA-induced
impairment of bile flow (57.4 ± 12.8 µl/50 min/g of liver,
n = 5, p < 0.05 versus TLCA; Fig. 2A). Wortmannin (100 nmol/liter) and TUDCA
additively counteracted TLCA-induced impairment of bile flow (83.9 ± 10.3 µl/50 min/g of liver, n = 5;
p < 0.05 versus control and TUDCA+TLCA; Fig. 2A). Thus, the PI3K inhibitor, wortmannin, did not
affect basal and TUDCA-induced bile flow but partly reversed
TLCA-induced inhibition of bile flow. Wortmannin and TUDCA exerted
additive anticholestatic effects.
Biliary HRP secretion was 0.88 ± 0.38 ng/min/g of liver
(n = 47) after loading of livers with HRP for 25 min
and a wash-out period of 5 min. HRP secretion slowly decreased during
the following 50 min (Fig. 1B) leading to a total secretion
of 19.5 ± 9.0 ng/50 min/g of liver (n = 5) in
control livers treated with Me2SO (0.1%, v/v) only (Fig.
1B). Wortmannin (100 nmol/liter) significantly increased
biliary HRP secretion (34.7 ± 8.1 ng/50 min/g of liver, p < 0.05, Fig. 1B). TLCA (10 µmol/liter)
markedly impaired biliary secretion of HRP (6.7 ± 2.3 ng/50 min/g
of liver, p < 0.05; Fig. 1B). Wortmannin
antagonized TLCA-induced impairment of HRP secretion both at low (100 nmol/liter) and high (500 nmol/liter) concentrations (22.1 ± 6.0 ng/50 min/g of liver n = 6 and 18.0 ± 5.5 ng/50
min/g of liver, n = 5, p < 0.01 versus TLCA; Fig. 1B).
Co-administration of wortmannin (100 nmol/liter) and TUDCA
(25 µmol/liter) led to a marked stimulation of biliary secretion of
HRP in normal livers (30.3 ± 3.0 ng/50 min/g of liver,
n = 5, p < 0.05, each,
versus control and TUDCA only; Fig. 2B) as well
as in livers treated with TLCA (10 µmol/liter) (46.6 ± 8.9 ng/50 min/g of liver, n = 5, p < 0.05 versus TLCA + TUDCA; Fig. 2B).
Thus, the PI3K inhibitor, wortmannin, stimulated biliary exocytosis
both under basal conditions and in the presence of TUDCA and completely
reversed TLCA-induced impairment of exocytosis. Again, wortmannin and
TUDCA exerted additive anticholestatic effects.
Organic Anion Secretion in Perfused Rat Livers (Protocol
II)--
Bile flow was 1.3 ± 0.2 µl/min/g of liver
(n = 40) after 20 min before bile acids or their
carrier Me2SO only (0.1%, v/v) were infused, indicating an
adequate secretory capacity of livers under these experimental
conditions. The addition of CDNB (30 µmol/liter for 10 min, min
41-50) led to a transient increase of bile flow under all experimental
conditions due to the choleretic potential of CDNB-glutathione
conjugate, GS-DNP, in rat liver. In controls, bile flow was 53.2 ± 7.7 µl/50 min/g of liver (n = 5) after CDNB
infusion. Wortmannin (100 nmol/liter) did not significantly affect bile
flow (92% of controls), whereas TLCA (10 µmol/liter) markedly
reduced bile flow to 6% of controls (Table I). Wortmannin (100 nmol/liter) induced a
marked increase of bile flow in TLCA-treated livers (33% of controls,
p < 0.01 versus TLCA; Table I). TUDCA (25 µmol/liter) increased bile flow to 177% of controls, and this
increase was not affected by concomitant treatment with wortmannin
(170% of controls; Table I). TUDCA (25 µmol/liter) reversed the
cholestatic effect of TLCA (10 µmol/liter) (128% of controls), and
wortmannin (100 nmol/liter) and TUDCA (25 µmol/liter) additively
counteracted the cholestatic effect of TLCA (10 µmol/liter) (176% of
controls, 137% of TUDCA + TLCA, Table I).
Thus, as in the first perfusion protocol, wortmannin did not affect
basal and TUDCA-induced bile flow but antagonized TLCA-induced impairment of bile flow. The anticholestatic effects of wortmannin and
TUDCA on TLCA-induced impairment of bile flow were additive and independent.
Biliary secretion of GS-DNP, a model Mrp2 substrate, was 730 ± 120 nmol/50 min/g of liver (n = 5; Table I) after
administration of CDNB (30 µmol/liter) for 10 min as described
previously (6). Wortmannin (100 nmol/liter) did not affect basal GS-DNP
secretion (94.7% of controls) (Table I). In contrast, TLCA (10 µmol/liter) markedly reduced GS-DNP secretion to 5.3% of controls
(Table I) as reported previously (6). Wortmannin (100 nmol/liter)
increased GS-DNP secretion in TLCA-treated livers from 5.3 to 15.0% of
controls (p < 0.05 versus TLCA). TUDCA (25 µmol/liter) stimulated GS-DNP secretion (127.0% of controls), and
this increase was not affected by concomitant treatment with wortmannin
(118.0% of controls). TUDCA (25 µmol/liter) largely antagonized the
effect of TLCA on secretion of GS-DNP (72.6% of controls,
p < 0.01 versus TLCA) as reported
previously (6), and wortmannin (100 nmol/liter) and TUDCA (25 µmol/liter) tended to additively counteract the effect of TLCA (10 µmol/liter) on GS-DNP secretion (82.5% of controls) (Table I).
Thus, wortmannin did not affect basal or TUDCA-induced secretion of the
model Mrp2 substrate, GS-DNP, but partly antagonized TLCA-induced
impairment of GS-DNP secretion. The anticholestatic effects of
wortmannin and TUDCA were independent.
Hepatovenous lactate dehydrogenase release after 85 min was not
affected by administration of wortmannin (100 nmol/liter) or TUDCA (25 µmol/liter). TLCA (10 µmol/liter) alone or in combination with
TUDCA (25 µmol/liter) markedly increased lactate dehydrogenase release (Table I). These effects were reversed by wortmannin (100 nmol/liter) (Table I). Thus, wortmannin did not induce liver cell
damage under the experimental conditions chosen but reversed liver cell
damage induced by TLCA alone or by TLCA and TUDCA.
Kinase Activities in Tissue of Perfused Rat Livers--
PI3K class
IA activity, as determined by a PI3K assay after immunoprecipitation
using an anti-PI3K p85 antibody, was reduced by wortmannin (100 nmol/liter) to 62% of controls (p < 0.01; Fig. 3). TLCA (10 µmol/liter) tended to
increase and TUDCA (25 µmol/liter) tended to decrease total PI3K
activity as determined by this methodological approach.
Immunoprecipitation of PI3K class 1A isoforms using PI3K p110
Thus, wortmannin inhibited PI3K activity in liver tissue. The limited
sensitivity of the methodological approach may have prevented
unequivocal disclosure of the effects of bile acids at low micromolar
concentrations on PI3K activity.
PI3K-dependent PKB (PKB/Akt) activity, a sensitive and
convenient physiological read-out of the activation of the PI3K pathway (27, 32) as determined by the amount of phospho-PKB(Ser-473) in liver
tissue, was reduced by wortmannin (100 nmol/liter) to 56% of controls
(p < 0.01, Fig. 4). TLCA
(10 µmol/liter) markedly stimulated PKB activity to 250%
(p < 0.001 versus control), and this effect
was completely reversed by wortmannin (100 nmol/liter; Fig. 4).
Interestingly, TUDCA (25 µmol/liter) reduced basal PKB activity to
54% of controls (p < 0.01, Fig. 4), and this effect was not further amplified by wortmannin (100 nmol/liter). In addition, TUDCA (25 µmol/liter) reduced TLCA-induced activation of PKB by 70%
(p < 0.01 versus TLCA, Fig. 4). The
addition of wortmannin (100 nmol/liter) led to an additional reduction
of PKB activity below control levels (p < 0.02 versus TLCA + TUDCA, Fig. 4).
Thus, TLCA markedly enhanced PKB activity, a sensitive marker of PI3K
activity, in liver tissue, whereas both wortmannin and TUDCA impaired
basal and TLCA-induced PKB activity in IPRL. The effects of wortmannin
and TUDCA on TLCA-induced PKB activity were additive and independent.
The
Thus, wortmannin did not affect membrane binding of PKC
PDK-1 activity in liver tissue (controls: 1.00 ± 0.26 AU,
n = 5) was not significantly affected by wortmannin
(100 nmol/liter; 0.78 ± 0.41 AU). Similarly, neither TLCA (10 µmol/liter) in the absence (0.98 ± 0.21 AU) or presence
(0.94 ± 0.50 AU) of wortmannin nor TUDCA (25 µmol/liter) in the
absence (1.04 ± 0.41 AU) or presence (1.05 ± 0.34 AU) of
wortmannin or TLCA + TUDCA in the absence (0.77 ± 0.61 AU) or
presence (1.00 ± 0.56 AU) of wortmannin affected PDK-1 activity
in liver tissue. Thus, PDK-1 activity was not altered by inhibition of
PI3K or bile acid administration under the experimental conditions chosen.
Secretion of Bile Acids by IRHC--
After incubation with the
fluorescent bile acid, CGamF (1 µmol/liter, 5 min), IRHC took up
1044 ± 648 AU (n = 7) CGamF. Wortmannin (100 nmol/liter) did not significantly affect uptake of CGamF (1493 ± 276 AU). Neither TLCA alone at 2.5 µmol/liter (971 ± 486 AU) or
5 µmol/liter (832 ± 208 AU) nor TLCA in the presence of wortmannin (100 nmol/liter) at 2.5 µmol/liter (1004 ± 348 AU) or 5 µmol/liter (1248 ± 606 AU) affected uptake of CGamF in
IRHC.
After 15 min, IRHC secreted 12.2 ± 3.9% (n = 7)
of CGamF into their canalicular space. Wortmannin (100 nmol/liter) did
not affect biliary secretion of CGamF (97% of controls, Fig.
6). TLCA (2.5 µmol/liter) markedly
reduced canalicular secretion of CGamF by 54% (p < 0.05; Fig. 6). Wortmannin completely reversed the effect of TLCA (106%
of controls, Fig. 6). Higher concentrations of TLCA (5 µmol/liter)
also impaired CGamF secretion (60% of controls), and wortmannin again
reversed the inhibiting effect of TLCA on canalicular secretion of
CGamF (93% of controls). Thus, wortmannin did not affect basal bile
acid secretion in IRHC but completely reversed the cholestatic effect
of TLCA on canalicular bile acid secretion.
PI3K-dependent PKB (PKB/Akt) Activity in
Isolated Rat Hepatocytes--
The amount of phospho-PKB(Ser-473), a
sensitive read-out of the activation of the PI3K pathway (27, 32), was
markedly enhanced by TLCA (5 µmol/liter) in hepatocytes in short term
culture (Fig. 7) and reached levels up to
194 ± 46% of controls after 60 min (p < 0.005 versus control; p < 0.05 versus
TUDCA; p < 0.01 versus TCA). In contrast,
TUDCA (10 µmol/liter) only transiently increased PKB activity,
whereas TCA (10 µmol/liter) had no effect under the experimental
conditions chosen (Fig. 7). Thus, TLCA markedly affected PI3K activity
in isolated hepatocytes in vitro, whereas TUDCA exerted only
minor transient effects on the PI3K pathway when administered at low
micromolar concentrations.
The present study indicates that the monohydroxy bile acid, TLCA,
impairs bile flow, hepatobiliary exocytosis, and secretion of bile
acids and other cholephiles by PI3K- and putatively
PKC TLCA was the first human bile acid identified to cause cholestasis and
jaundice (1), yet the molecular mechanisms by which TLCA induces
cholestasis have remained obscure. TLCA induces selective damage of
canalicular membranes leading to an increase in membrane rigidity and
loss of microvilli (33, 34). TLCA impairs transcellular movement of
vesicles (35) as well as vesicle fusion at the apical pole (6) and
inhibits secretion of organic anions and bile acids across the
canalicular membrane (6, 7, 36). The recent finding that TLCA markedly
reduces the density of the conjugate export pump, Mrp2, in the
canalicular membrane (6) strongly supports the concept that the
mechanism of TLCA-induced cholestasis involves inhibition of
vesicle-mediated carrier insertion in the apical liver cell membrane.
This view is further supported by the present study demonstrating that
the PI3K inhibitor, wortmannin, completely reverses TLCA-induced
inhibition of hepatobiliary exocytosis in IPRL in vivo as
well as canalicular bile acid secretion in IRHC in
vitro.
Effects of the PI3K inhibitor, wortmannin, and of bile acids on total
activity of class IA PI3K were determined in IPRL in the
present study. Class IA PI3K are assumed to represent a
predominant form of PI3K in secretory cells. Wortmannin inhibited PI3K
activity in IPRL (Fig. 3), confirming that the effects of wortmannin on TLCA-induced cholestasis were mediated by PI3K in the present study.
Bile acids at low micromolar concentrations did not induce significant
changes of total class IA PI3K activity as determined by a
PI3K assay in IPRL (Fig. 3), although the PI3K inhibitor, wortmannin,
markedly affected TLCA-induced changes of secretion (Figs. 1 and 2).
Thus, we speculate that low micromolar concentrations of bile acids may
modulate PI3K activity in hepatocytes in vivo at a
subcellular level that is not technically detectable when using a PI3K
assay in liver homogenates.
The serine/threonine protein kinase Akt/PKB is a well characterized
target and effector of PI3K (13) and is used as a sensitive read-out of
PI3K activity (27, 32). Binding of lipid products of PI3K to the PKB
pleckstrin homology domain is critical for PKB activation via
phosphoinositide-dependent kinase-1 (PDK-1)-mediated phosphorylation (13). Accordingly, the specific PI3K inhibitor, wortmannin, reduced basal PKB activity in liver tissue in the present
study (Fig. 4). Our new finding that the cholestatic bile acid TLCA and
the anticholestatic bile acid TUDCA inversely regulate PKB activity in
IPRL at low micromolar concentrations is of interest. TLCA-induced
activation of PKB was completely reversed by wortmannin, further
supporting activation of a PI3K-dependent signaling pathway by TLCA (Fig. 4). In contrast, TUDCA impaired PKB activity both under
basal conditions and in livers treated with TLCA. The finding that
wortmannin did not affect TUDCA-induced impairment of PKB activity
under basal conditions and reversed TLCA-induced activation of PKB by
effects that were additive to TUDCA supports the concept that TUDCA
exerted its anticholestatic effects in the present model in a
PI3K-independent manner, whereas cholestatic effects of TLCA were
mediated by PI3K-dependent mechanisms. The exact molecular
mechanisms by which bile acids inversely regulate PI3K and PKB activity
remain to be elucidated.
TLCA has already previously been shown to affect hepatocellular
signaling cascades, which control vesicular exocytosis and membrane
protein targeting in secretory cells. (i) TLCA specifically induces
translocation of the The Interestingly, wortmannin and TUDCA exerted additive and independent
anticholestatic effects on bile flow and organic anion secretion as
well as hepatobiliary exocytosis in TLCA-treated IPRL in the present
study. Submaximal dosing of wortmannin was virtually excluded as a
potential explanation for these additive effects of wortmannin and
TUDCA because administration of the PI3K inhibitor at doses of 100 and
500 nmol/liter resulted in comparable effects on TLCA-induced
impairment of bile flow in IPRL (Fig. 1a). As shown
previously, the anticholestatic effect of TUDCA on TLCA-induced
impairment of organic anion secretion (and bile
flow)2 was mediated by
PKC In the present study wortmannin did not affect basal bile flow but
stimulated biliary exocytosis in IPRL preloaded with HRP. These
findings are in contrast to a study by Folli et al. (42) who
show that wortmannin reduces bile flow and biliary HRP secretion in
IPRL. However, the experimental protocols of the two studies differed.
Folli et al. (42) investigated the effect of wortmannin on
hepatic uptake (endocytosis), transcellular trafficking, and biliary
excretion of HRP in IPRL (42), whereas the present study mainly focused
on the role of PI3K in exocytosis. In the previous study, inhibition of
PI3K impaired endo- and transcytosis of fluid phase markers in IRHC and
may, therefore, have impaired HRP uptake and transport across the
hepatocyte in IPRL. In the present study, HRP was endocytosed before
administration of wortmannin. Thus, in livers preloaded with HRP,
stimulation of exocytosis by wortmannin may have antagonized a weak
inhibiting effect of wortmannin on total bile flow, although the
vesicular pathway may contribute less than 10% to total bile flow in
IPRL (43). Altogether, the findings of these two studies suggest that
basolateral endocytosis is stimulated, and apical exocytosis is
suppressed by intrinsic PI3K activity in IPRL.
PI3K has also recently been demonstrated to be involved in regulation
of canalicular bile acid secretion. Misra et al. (14, 15)
observed that secretion of TCA by IPRL is mediated in part via
PI3K-dependent mechanisms. Transport of TCA across the
canalicular membrane was markedly reduced by wortmannin in IPRL and
canalicular membrane vesicles. In contrast, the present study indicates
that TLCA-induced impairment of bile acid secretion (Fig. 6) as well as
bile flow, exocytosis, and organic anion secretion (Fig. 1, Table I) is
reversed by wortmannin. Can these differences be explained? Different
classes and subclasses of PI3K have been described that are all
inhibited by the PI3K inhibitor, wortmannin (44). Class I PI3K are
heterodimers made up of a 110-kDa catalytic subunit (p110) and an
adaptor/regulatory subunit. Three p110 isoforms ( TUDCA has been shown to stimulate TCA secretion in normal IPRL in part
by a PI3K-dependent mechanism and to stimulate PI3K activity at least transiently in isolated hepatocytes when administered at 500 µmol/liter (23). The present study confirmed transient stimulation of PI3K by TUDCA at 10 µmol/liter in isolated hepatocytes as determined by phosphorylation of PI3K-dependent PKB
(Fig. 7). However, the present study did not reveal a role of PI3K in
mediating choleretic and anticholestatic effects of TUDCA in
vivo; bile flow, exocytosis, and organic anion secretion in IPRLs
treated with TUDCA were not affected by wortmannin (Fig. 2, Table I). In addition, the anticholestatic effects of TUDCA in TLCA-treated livers were even enhanced when wortmannin was co-administered (Fig. 2).
Thus, a mediator function of PI3K in TUDCA-induced bile secretion may
be restricted to secretion of bile acids in normal liver.
In the present study, co-administration of a PI3K inhibitor not only
reversed TLCA-induced impairment of bile secretion but also cellular
damage as determined by lactate dehydrogenase release (Table I). The
improvement in bile flow alone could not account for this effect since
TUDCA also improved secretion in TLCA-treated livers but failed to
abolish the cell damage induced by TLCA in IPRL. Future studies will be
necessary to elucidate the role of PI3K in TLCA-induced acute liver
cell damage.
The present data suggest that PI3K represents a potential target of
future anticholestatic treatment strategies. It should be mentioned,
however, that PI3K may activate a survival pathway in rat hepatocytes
treated with the hydrophobic bile acid, taurochenodeoxycholic acid
(TCDCA) which protects liver cells from TCDCA-induced damage in
vitro (45) as well as in vivo (Rust C, unpublished
observation). Interestingly, the taurochenodeoxycholic acid-induced
survival pathway did not involve PKB activation in vitro
(45). Thus, different bile acids may exert differential effects on
PI3K- and PKB-mediated processes in liver cells. It remains to be
clarified whether involvement of different PI3K isoforms or action in
different subcellular compartments may contribute to these diverse
effects of bile acids on PI3K and PKB.
In summary, the present study demonstrates that TLCA-induced impairment
of bile flow, hepatobiliary exocytosis, secretion of bile acids, and
other organic anions as well as liver cell damage is mediated by PI3K-
and putatively PKC
(PKC
)-dependent mechanisms. Products of
phosphatidylinositol 3-kinases (PI3K) stimulate PKC
. We studied the
role of PI3K for TLCA-induced cholestasis in isolated perfused rat
liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA
(10 µmol/liter) impaired bile flow by 51%, biliary secretion of
horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and
the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by
95% and stimulated PI3K-dependent protein kinase B, a
marker of PI3K activity, by 154% and PKC
membrane binding by 23%.
In IRHC, TLCA (2.5 µmol/liter) impaired canalicular secretion of the
fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The
selective PI3K inhibitor, wortmannin (100 nmol/liter), and the
anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 µmol/liter) independently and additively reversed the effects of TLCA
on bile flow, exocytosis, organic anion secretion,
PI3K-dependent protein kinase B activity, and PKC
membrane binding in IPRL. Wortmannin also reversed impaired bile acid
secretion in IRHC. These data strongly suggest that TLCA exerts
cholestatic effects by PI3K- and PKC
-dependent
mechanisms that are reversed by tauroursodeoxycholic acid in a
PI3K-independent way.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-isoform of protein kinase
C (PKC
), a mediator of regulated exocytosis (6), and (iii)
selectively translocate the Ca2+-independent
-isoform of
PKC to canalicular membranes and activate membrane-bound PKC (6, 11).
The role of PKC
as a mediator of TLCA-induced cholestasis, however,
remains elusive because specific PKC
inhibitors for in
vivo use are not available.
-isoform of PKC in transfected insect cells as well as in human
hepatoma cells (16, 17), possibly via binding and recruitment to
membranes of phosphoinositide-dependent kinase-1 (PDK-1)
(18) and subsequent PDK-1-dependent phosphorylation and
autophosphorylation of PKC
(19) in a way similar to activation of
the best characterized PI3K effector, the proto-oncogene Akt/protein kinase B (PKB) (13). Therefore, we speculate that the TLCA cholestatic effects may be mediated by PI3K- and PKC
-dependent
mechanisms. PI3K can be selectively blocked by specific PI3K
inhibitors, among which wortmannin is the best characterized in
vivo and in vitro (20).
-dependent mechanisms (6). Recently, PI3K was
proposed to contribute to TUDCA-induced stimulation of bile acid
secretion in normal rat liver (23).
in
TLCA-induced impairment of bile secretion in vivo in the model of the isolated perfused rat liver (IPRL) as well as in vitro in isolated rat hepatocyte couplets (IRHC) using the
selective PI3K inhibitor wortmannin. We also investigated the role of
PI3K in the ability of TUDCA to reverse TLCA-induced impairment of bile
flow, organic anion secretion, and hepatobiliary exocytosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and p110
antibodies
and a PDK-1 immunoprecipitation kinase assay kit were from Upstate
Biotechnology (Lake Placid, NY). Rabbit anti-peptide antibodies
anti-PKC
, fetal calf serum, Geneticin, and trypsin/EDTA were
purchased from Invitrogen. Anti-phospho PKB (Ser-473) and anti-PKB
antibodies were from Cell Signaling (Beverly, MA). Molecular weight
markers and protein A/G plus-agarose were from Santa Cruz Biotechnology
(Santa Cruz, CA), and Hyperfilm ECL was from Amersham Biosciences.
Immobilon-P membranes were from Millipore (Eschborn, Germany). A goat
anti-rabbit IgG antibody was from Bio-Rad. Aprotinin,
taurine-conjugated bile acids, dimethyl sulfoxide (Me2SO),
horseradish peroxidase (HRP, type II), leupeptin, wortmannin, and
albumin (fraction V) were from Sigma. 1-Chloro-2,4-dinitrobenzene
(CDNB) was from ICN Biomedicals Inc. (Aurora, OH). Easytides
[
32P]ATP was from PerkinElmer Life Sciences. A
renaissance Western blot chemiluminescence reagent was from PerkinElmer
Life Sciences. All other chemicals were of the highest purity
commercially available.
distribution (see below).
80 °C for
determination of PI3K activity and PKB (Ser-473) phosphorylation (see below).
dVbile (E335, absorption at 335 nm;
Vtotal, 1005 µl;
, molar absorption coefficient 9.6 liters nmol
1 cm
1;
d, 1 cm; Vbile, 5 µl). The low
background absorption at 335 nm of the bile sample collected just
before infusion of CDNB was set as 0.
, or p110
antibody at
4 °C overnight and precipitated with 80 µl of A/G-agarose for 2-4
h at 4 °C. After centrifugation (20,000 × g, 1 min,
4 °C), the pellet was washed 3 times with lysis buffer, 3 times with
buffer B (Tris, 100 mmol/liter; LiCl, 5 mmol/liter;
Na3VO4, 0.1 mmol/liter; pH 7.4, 4 °C) and 2 times with buffer C (NaCl, 150 mmol/liter; Tris, 10 mmol/liter; EDTA, 5 mmol/liter; Na3VO4, 0.1 mmol/liter; pH 7.4, 4 °C). The PI3K assay was then started by adding 50 µl of a
reaction buffer (Tris, 10 mmol/liter; MgSO4, 20 mmol/liter; DL-dithiothreitol, 2 mmol/liter; pH 7.5, 37 °C)
supplemented with 20 µg of PtdIns, 10 µCi of
[
-32P]ATP, and 200 µmol/liter ATP, and samples were
incubated at 37 °C for 10 min. The reaction was stopped by adding
150 µl of chloroform:methanol:HCl, 12 mol/liter (33:66:0.6). For
lipid extraction, 120 µl of chloroform were added, and samples were
centrifuged (20,000 × g for 10 min at 4 °C). The
organic phase was washed by adding 150 µl of CH3OH/HCl, 1 mol/liter (1:1), 20 µl of HCl, 8 mol/liter, and 160 µl of
CHCl3/CH3OH (1:1). After centrifugation
(20,000 × g, 10 min, 4 °C) the organic phase was
evaporated under N2 and spotted on TLC plates. Plates were
developed in CHCl3, CH3OH, H2O,
25% NH3 (60:47:11.3:2). Autoradiography and densitometry
of spots representing the PI3K product, phosphatidylinositol 3-phosphate, allowed calculation of PI3K activity.
80 °C) was
homogenized in ice-cold lysis buffer (27) (1 ml/100 mg of tissue) and
processed as described (27, 28). Aliquots were electrophoresed using
sodium dodecyl sulfate, 10% polyacrylamide gel electrophoresis.
Separated proteins were transferred to Immobilon-P membranes and probed
with phospho-PKB (AktSer-473) antibodies at a dilution of
1:1000 overnight to detect the activated form of PKB. Then membranes
were stripped and reprobed with a PKB antibody (1:1000) to detect total
PKB in an identical procedure. After the use of a goat anti-rabbit IgG
antibody (1:5000), a chemiluminescence reagent, and Hyperfilm ECL, the
phospho-PKB and PKB bands were quantified by densitometry (NIH Image
Densitometric Analysis 1.54; Bethesda, MD, 1994).
in hepatic tissue was
determined by an immunoblotting technique exactly as described
previously (6, 11, 29) using affinity-purified isoenzyme-specific
antibodies for PKC
. The PKC bands at 90 kDa (
) were quantified by
densitometry (NIH Image Densitometric Analysis 1.54; Bethesda, MD,
1994). Results were expressed as [(optical density of the particular
fraction)/(total optical density of cytosol and membrane fraction)] × 100 (%).
-glycerophosphate, 10 mmol/liter; Na4P2O7, 5 mmol/liter;
Na3VO4, 1 mmol/liter; 2-mercaptoethanol, 0.1%;
H2O, 50 ml; pH 7.5, 4 °C) and further processed exactly following the assay protocol provided by the manufacturer. After immunoprecipitation of PDK-1 with anti-PDK-1 sheep
immunoaffinity-purified IgG and protein G-agarose beads, PDK-1 activity
was determined by phosphorylation and activation of recombinant serum
and glucocorticoid-inducible kinase 1 (SGK1) and
SGK1-dependent phosphorylation of an Akt/SGK substrate
peptide using [
-32P]ATP. The phosphorylated substrate
peptide was separated from residual [
-32P]ATP using
P81 phosphocellulose paper and quantitated using a scintillation
counter. PDK-1 activity was expressed in arbitrary units (AU). Mean
PDK-1 activity in control livers was set as 1.00 AU.
80 °C). Shock-frozen cells were homogenized in ice-cold lysis
buffer (27) (1 ml/100 mg) and processed as described above (27, 28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The PI3K inhibitor, wortmannin (100 nmol/liter), reverses TLCA-induced impairment of bile flow
(A) and biliary secretion (B) of HRP
in the isolated perfused rat liver (for experimental details, see
"Experimental Procedures," "Protocol I"). Livers were
preloaded with HRP. Then wortmannin (Wo; 100 nmol/liter;
), TLCA (10 µmol/liter;
), wortmannin (100 nmol/liter) + TLCA
(10 µmol/liter;
), wortmannin (500 nmol/liter) + TLCA (10 µmol/liter; x) or Me2SO only (0.1%, v/v; control;
)
were administered for 50 min. Results are expressed as the mean ± S.D. of 5-6 experiments. For statistical evaluation, see
text.
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Fig. 2.
The PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid, TUDCA, additively
counteract TLCA-induced impairment of bile flow (A)
and biliary secretion (B) of HRP in the isolated
perfused rat liver (for experimental details, see "Experimental
Procedures," "Protocol I"). Livers were preloaded with HRP.
After a wash-out phase of 5 min, TUDCA (25 µmol/liter; ), TUDCA
(25 µmol/liter) + wortmannin (Wo; 100 nmol/liter;
),
TUDCA (25 µmol/liter) + TLCA (10 µmol/liter;
), and TUDCA (25 µmol/liter) + wortmannin (100 nmol/liter) + TLCA (10 µmol/liter;
) were administered for 50 min. Results are expressed as the
mean ± S.D. of 5-6 experiments. For statistical
evaluation, see "Results."
Effect of the PI3K inhibitor, wortmannin (100 nmol/liter), on bile
flow, biliary secretion of the model Mrp2 substrate, GS-DNP, and
lactate dehydrogenase (LDH) release into the hepatovenous effluate in
isolated perfused rat liver (for experimental details, see
"Experimental Procedures," "Protocol II")
or
p110
antibodies revealed similar results (data not shown).
View larger version (29K):
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Fig. 3.
PI3K activity in liver tissue in the absence
(white bars) or presence (black bars)
of the PI3K inhibitor, wortmannin (Wo, 100 nmol/liter), under the experimental conditions described in Table
I. PI3K activity was determined in shock-frozen liver tissue after
immunoprecipitation using an anti-PI3K p85 antibody as described under
"Experimental Procedures." The product of PI3K,
phosphatidylinositol 3-phosphate (PtdIns(3)P), was
identified by TLC and autoradiography. The bar graph
represents the amount of phosphatidylinositol 3-phosphate formed by
immunoprecipitates of liver tissue from experiments shown in Table I as
quantitated by phosphorimaging analysis. Immunoprecipitation with
anti-PI3K p110 and p110
antibodies revealed similar results.
Results are given as the mean ± S.D. of four experiments each.
DMSO, Me2SO. *, p < 0.01 versus control; #, p < 0.05 versus TLCA.
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Fig. 4.
TLCA-induced activation of
PI3K-dependent PKB is reversed by the PI3K inhibitor,
wortmannin (Wo, 100 nmol/liter), and the
anticholestatic bile acid, TUDCA, in liver tissue under the
experimental conditions described in Table I. PKB activity was
determined in shock-frozen liver tissue as the amount of pPKB(Ser-473)
using a specific pPKB(Ser-473) antibody and a Western blotting
technique as described under "Experimental Procedures." In
parallel, total PKB mass was determined on each blot using a
nonselective PKB antibody to prove that the total amount of PKB was
identical on each lane. Panel A shows
representative immunoblots of which the upper bands
represent pPKB(Ser-473), and the lower bands represent total
PKB under different experimental conditions. The bar graph
in B represents activated PKB as determined by the amount of
pPKB(Ser-473) in liver tissue from experiments shown in Table I.
Results are given as mean ± S.D. of five experiments each. *,
p < 0.05 versus control. DMSO,
Me2SO.
-isoenzyme of PKC was about equally distributed between cytosol
(57.4 ± 5.8%, n = 5) and membranes (42.6 ± 5.8%) in control livers treated with Me2SO. Neither
wortmannin (100 nmol/liter) nor TUDCA (25 µmol/liter) affected
distribution of PKC
(Fig. 5). In
contrast, TLCA (10 µmol/liter) significantly increased membrane
binding of PKC
by 23.0% (p < 0.05) as observed
previously in isolated hepatocytes (11). Wortmannin (100 nmol/liter) as well as TUDCA (25 µmol/liter) reversed the effect of TLCA on PKC
membrane binding (Fig. 5). Interestingly, wortmannin significantly reduced PKC
membrane binding by 28% also in livers treated with TLCA and TUDCA (Fig. 5).
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Fig. 5.
TLCA-induced PKC
membrane binding is reversed by the PI3K inhibitor, wortmannin
(100 nmol/liter), and the anticholestatic bile acid, TUDCA, in liver
tissue under the experimental conditions described in Figs.
1 and 2. PKC
membrane binding was determined by a Western
blotting technique as described under "Experimental Procedures."
Results are given as the mean ± S.D. of 5 or 6 experiments each.
*, p < 0.05 versus control; #,
p < 0.05 versus TLCA + TUDCA.
DMSO, Me2SO.
in liver
tissue under basal conditions, but like TUDCA, reversed TLCA-induced
membrane binding of PKC
. The effects of wortmannin and TUDCA on
TLCA-induced membrane binding of PKC
were independent and additive.
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Fig. 6.
The PI3K inhibitor, wortmannin (black
bars), reverses TLCA-induced impairment of canalicular
secretion of the fluorescent bile acid CGamF in IRHC (for experimental
details, see "Experimental Procedures"). IRHC were pretreated
for 10 min with Me2SO only (0.1%, v/v; control),
wortmannin (100 nmol/liter), TLCA (2.5 µmol/liter), or wortmannin
(100 nmol/liter) + TLCA (2.5 µmol/liter). IRHC were then incubated
for 5 min with CGamF (1 µmol/liter) and again treated for 15 min with
the agonists mentioned above. Results are given as the mean ± S.D. of 5-7 experiments from 7 different preparations. *,
p < 0.05 versus control. DMSO,
Me2SO.
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Fig. 7.
The hydrophobic bile acid TLCA (5 µmol/liter) induces sustained activation of
PI3K-dependent PKB, a sensitive read-out of PI3K activity,
in isolated rat hepatocytes in short term culture, whereas TUDCA
(10 µmol/liter) induces transient activation,
and TCA (10 µmol/liter) is ineffective under
the experimental conditions chosen. Results are given as the
mean ± S.D. of seven-eight experiments from eight different
preparations. *, p < 0.05 versus control;
**, p < 0.005 versus control; §,
p < 0.05 versus TLCA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dependent mechanisms. The major finding of this
study is that TLCA-induced cholestasis can be reversed by specific PI3K
inhibitors. This is demonstrated by the reversal of TLCA-induced
impairment of bile flow and HRP secretion in IPRL (Figs. 1 and 2) as
well as the reversal of TLCA-induced impairment of CGamF secretion in IRHC (Fig. 6) after administration of wortmannin. Thus, this study confirms that an individual bile acid can modulate liver cell function
including bile secretion by interacting with specific signal
transduction pathways in hepatocytes.
-isoform of PKC to the canalicular membrane,
increases intracellular levels of the physiological PKC activator,
sn-1,2-diacylglycerol, and activates membrane-bound PKC (11,
29). (ii) TLCA modulates [Ca2+]i (cytosolic free
calcium) in isolated hepatocytes (8-10, 37, 38) and may inhibit
Ca2+ influx in vitro at concentrations
10
µmol/liter (8-10). Both, activation of PKC
and impairment of
Ca2+ influx have been related to impairment of exocytosis
and membrane targeting of proteins (39, 40).
-isoform of PKC is specifically activated in vitro by
products of PI3K, PtdIns-3,4-bisphosphate and
PtdIns-3,4,5-trisphosphate (16, 17), possibly via
phosphoinositide-dependent kinase I (PDK-1)-induced
phosphorylation of Thr-566 in the activation loop and subsequent
autophosphorylation of Ser-729 in the C-terminal hydrophobic motif
(19). PDK-1 activity was not affected by wortmannin and bile acids in
the present study (see "Results"). In human HepG2 hepatoma cells,
activation of PI3K via stimulation of a mutant platelet-derived growth
factor receptor led to specific translocation of PKC
from cytosol to
membranes, a key step for activation of PKC
. This process was
reversed by the addition of the PI3K inhibitor, wortmannin (17). The
in vivo findings in the present study are consistent with
these observations. TLCA-induced translocation of PKC
to membranes
was reversed by wortmannin and, as recently shown, by the
anticholestatic bile acid TUDCA (6). PKC
membrane binding was even
more strongly inhibited when wortmannin was co-administered with TUDCA
(Fig. 5). Thus, TLCA-induced membrane translocation of PKC
seems to
be mediated by PI3K-dependent mechanisms.
- and putatively Ca2+-dependent
mechanisms (6) as documented by reversal of the anticholestatic effect
of TUDCA by use of the PKC inhibitor, bisindolylmaleimide I. Bisindolylmaleimide I predominantly blocks the
Ca2+-sensitive
-isoform of PKC. PKC
is selectively
translocated by TUDCA to hepatocellular membranes (29, 41). TLCA
impaired membrane binding of the Ca2+-sensitive PKC
(6),
whereas TUDCA reversed TLCA-induced impairment of PKC
membrane
binding (6). Thus, we speculate that TLCA may impair targeting of
apical carrier proteins and, thereby, hepatobiliary secretion by a dual
mechanism that includes activation of PI3K and, subsequently, PKC
at
the apical pole of the hepatocyte on one hand and impairment of
Ca2+ influx and PKC
membrane binding on the other hand.
Further work is needed to corroborate this assumption.
,
,
) and at
least seven adaptor proteins (p85, p55) may form class IA
PI3K family members. In contrast, class IB PI3K
(p110
/p101) are only abundant in mammalian white blood cells. PtdIns
4,5-bisphosphate appears to be the preferred substrate of class I PI3K
in vivo, although these PI3K can also utilize PtdIns and
PtdIns 4-phosphate as substrates in vitro (44). Three class
II isoforms (PI3K-C2
, -
, -
) have been detected in mammalian
tissue. Their molecular mass is above 170 kDa, and their preferred
substrate is PtdIns 4-phosphate. The
-isoform is mainly detected in
liver (44). Class III PI3K are homologues of yeast vesicular-sorting
protein Vsp34p and use only PtdIns as substrate (44). As cellular
levels of PtdIns 3-phosphate are quite constant under physiological
conditions, their role in short-term regulation of cellular metabolism
is regarded as limited. Thus, it appears possible that different bile
acids such as TCA or TLCA affect different subclasses of PI3K that are
involved in regulation of biliary secretion. Future development of
specific inhibitors may permit differentiation of the actions of these
different PI3K isoforms.
-dependent mechanisms. TUDCA reversed
the inhibitory effects of TLCA on bile secretion by a PI3K-independent mechanism.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grants Be 1242/5-3 and Be 1242/5-4 (to U. B.) and in part by National Institutes of Health Grants DK 25636 (to J. L. B.) and DK P30-34989 (to the Yale Liver Center). Some of the data were presented at the 36th Annual Meeting of the European Association for the Study of the Liver, Prague, Czechia, April 19-22, 2001 and at the 51st Annual Meeting of the American Association for the Study of Liver Disease, Dallas, TX, November 9-13, 2001 and were published in part in abstract form (Denk, G. U., Wimmer, R., Rust, C., Paumgartner, G., and Beuers, U. (2001) J. Hepatol. 34, Suppl. 1, 187 (abstr.) and Beuers, U., Denk, G. U., Soroka, C. J., Wimmer, R., Rust, C., Paumgartner, G., and Boyer, J. L. (2001) Hepatology 34, 471 (abstr.)).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.
§ Both authors contributed equally to this work.
¶ To whom correspondence should be addressed: Dept. of Medicine II, Grosshadern, Klinikum of the University of Munich, Marchioninistrasse 15, D-81377 Munich, Germany. Tel.: 49-89-7095-2272; Fax: 49-89-7095-5271; E-mail: beuers@med2.med.uni-muenchen.de.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M209898200
2 U. Beuers, G. U. Denk, and R. Wimmer, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: TLCA, taurolithocholic acid; UDCA, ursodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; IRHC, isolated rat hepatocyte couplets; AU, arbitrary units; CDNB, 1-chloro-2,4-dinitrobenzene; CGamF, cholylglycylamido fluorescein; Me2SO, dimethyl sulfoxide; GS-DNP, 2,4-dinitro-S-glutathione; HRP, horseradish peroxidase; Mrp2, rat conjugate export pump; PDK-1, phosphoinositide-dependent kinase-1; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PKC, protein kinase C; TCA, taurocholic acid; TLC, thin layer chromatography; IPRL, isolated perfused rat liver; PtdIns, phosphatidylinositol.
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REFERENCES |
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