1 Medical Physiology, 2 Department of Internal Medicine, 3 R & E, Scott & White Hospital, The Texas A&M University System Health Sciences Center, College of Medicine, and 8 Central Texas Veterans Health Care System, Temple, Texas 76504; 4 Division of Gastroenterology, Tohoku University School of Medicine, 1 - 1 Seiryo, Aobaku, Sendai, Japan 980 - 8574; 6 Department of Gastroenterology, University of Ancona, Ancona 60100; 5 Division of Gastroenterology, University of Rome, "La Sapienza," Rome 00185; and the 7 Department of Public Health, University of Rome Tor Vergata, Rome, Italy 00185
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of this study was to determine whether taurocholate prevents vagotomy-induced cholangiocyte apoptosis. After bile duct ligation (BDL) + vagotomy, rats were fed taurocholate for 1 wk in the absence or presence of wortmannin. Caspase involvement was evaluated by measurement of caspase 8, 9, and 3 activities. Proliferation was determined by morphometry and PCNA immunoblots. Changes in phosphatidylinositol 3-kinase (PI3-kinase) activity were estimated by the expression of the phosphorylated Akt protein. Apically located Na+-dependent bile acid transporter (ABAT) expression and activity were evaluated by immunoblots and [3H]taurocholate uptake, respectively. Cholangiocyte apoptosis increased, whereas proliferation decreased in BDL + vagotomy rats. Taurocholate feeding prevented vagotomy effects on cholangiocyte functions, which were abolished by wortmannin. ABAT expression and activity as well as phosphorylated Akt protein expression were reduced by vagotomy but restored by taurocholate. The activities of caspase 8, 9, and 3 increased in BDL + vagotomy rats but were restored by taurocholate. The protective effect of taurocholate was associated with maintenance of ABAT activity, downregulation of caspase 8, 9, and 3, and activation of PI3-kinase. Bile acids are important in modulating cholangiocyte proliferation in denervated livers.
apoptosis; bile flow; intrahepatic biliary epithelium; proliferation; secretin
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CHOLANGIOCYTES ARE THE TARGET cells in several human cholestatic liver diseases (i.e., cholangiopathies), characterized by cholangiocyte proliferation and/or ductopenia (9). Cholangiocyte proliferation is involved, as a repair mechanism, in counteracting loss of bile ducts, thereby inhibiting the progression of cholangiopathies toward the final ductopenic stage (9). In animal models, activation of proliferation of cholangiocytes (which are normally mitotically dormant) (30) is triggered by a number of pathological events including bile duct ligation (BDL) (6, 8, 17) or feeding of certain bile acids (7, 10). Cholangiocyte loss can be achieved by acute administration of CCl4 (31, 32). Although cholangiocyte proliferation is coupled with increased secretin-stimulated choleresis (6, 8, 18, 32), ductopenia is associated with decreased secretin-induced ductal secretion (32).
We have previously shown (28) that interruption of the parasympathetic innervation by total vagotomy inhibits cholangiocyte proliferation in BDL rats and induces activation of cholangiocyte apoptosis leading to ductopenia. Vagotomy-induced cholangiocyte apoptosis and loss of proliferation are linked to reduced intracellular cholangiocyte cAMP levels, because maintenance of cAMP levels, by administration of forskolin, prevented the effects of vagotomy on cholangiocyte growth/ductopenia (28). The serum levels of gastrin, somatostatin, and insulin were unaffected by vagotomy in BDL rats, thus supporting the concept that our findings were not caused by changes in the release in the blood of these hormones, but rather by a direct effect of the cholinergic system on the intrahepatic biliary epithelium (28).
Bile acids enter cholangiocytes through the apically located Na+-dependent bile acid transporter (ABAT) (5, 27). After uptake, bile acids modulate cholangiocyte secretion, proliferation, and apoptosis (4, 7, 10). Bile salts affect cell functions through the phosphatidylinositol 3-kinase (PI3-kinase) pathway (35, 45, 52). For example, the hydrophobic bile acid taurochenodeoxycholate activates PI3-kinase and inhibits caspase 8 activity in hepatocytes (45, 49), which prevent its inherent toxicity (45).
cAMP protects cultured rat hepatocytes from apoptosis in a PI3-kinase-dependent manner (52, 53). In previous studies (2-4, 7), we have shown that taurocholate increases both basal and secretin-stimulated intracellular cAMP levels in cholangiocytes, both in vitro and in vivo, when given to rats by a dietary regimen containing this bile acid.
We posed the following questions: first, does taurocholate feeding prevent vagotomy-induced apoptosis of cholangiocytes, vagotomy inhibition of cholangiocyte proliferation, and secretin-stimulated ductal secretion? Second, are taurocholate effects on vagotomy-induced bile duct damage mediated by the PI3-kinase/Akt pathway? And third, are changes in cholangiocyte apoptosis, proliferation, and secretion (following vagotomy and taurocholate feeding) dependent on ABAT activity?
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Reagents were purchased from Sigma (St. Louis, MO) unless otherwise
indicated. Rat chow, containing 1% taurocholate or control diet
(AIN-76), was prepared from Dyets (Bethlehem, PA). Control chow
(AIN-76) has the same composition of the chow containing 1%
taurocholate, but it does not contain taurocholate. The substrate for
-glutamyltranspeptidase (
-GT),
N-(
-L-glutamyl)-4-methoxy-2-naphthylamide, was purchased from Polysciences (Warrington, PA). The PKA inhibitor, Rp-cAMP (12), and the caspase 8 inhibitor, Z-IETD-fmk
(44), were purchased from Calbiochem (San Diego, CA). RIA
kits for the determination of intracellular cAMP levels were purchased
from Amersham (Arlington Heights, IL). [3H]taurocholate
(3.47 Ci/mmol) was purchased from New England Nuclear (Boston, MA). The
antibodies vs. total and phosphorylated Akt (Ser473) were
purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The
antibody vs. ABAT (rabbit anti-rat ABAT) was a gift from Dr. P. Dawson
(Bowman Gray School of Medicine, Winston-Salem, NC).
Animal models. Male Fischer 344 rats (150-175 g) were purchased from Charles River (Wilmington, MA), kept in a temperature-controlled environment (20-22°C) with a 12:12-h light-dark cycle, and fed ad libitum with the selected diet. The majority of the studies were performed in the following four groups of animals: 1) rats that, immediately after BDL (for cell isolation) (1, 3, 6-8, 10, 17, 24, 28, 29, 32) or bile duct incannulation (BDI; for bile collection) (8), were fed control diet or 1% taurocholate for 1 wk; and 2) rats that (immediately after BDL or BDI) underwent vagotomy and, subsequently, were fed control diet or 1% taurocholate diet for 1 wk.
To demonstrate the link between cholangiocyte apoptosis and the PI3-kinase-signaling pathway, rats, immediately after BDL + vagotomy, were fed taurocholate and subsequently treated with wortmannin, a specific PI3-kinase inhibitor (35) (1 daily intraperitoneal injection of 0.7 mg kg/body wt) (38) in DMSO for 1 wk. To demonstrate the link between cholangiocyte apoptosis and the ERK-signaling pathway, rats, immediately after BDL + vagotomy, were fed taurocholate and subsequently treated with 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U-0126; an MEK inhibitor, 1 daily intraperitoneal injection of 5 mg kg/body wt) (14, 36) in DMSO for 1 wk. To demonstrate whether vagotomy-induced cholangiocyte apoptosis is mediated through the extrinsic or intrinsic apoptotic pathway, rats, immediately after BDL + vagotomy, were treated with Z-IETD-fmk [a specific caspase 8 inhibitor (44), by daily intraperitoneal injections, 4.6 µg · kg body wtAssessment of biliary bile acid concentration and composition.
The concentration of total bile salts in bile from rats that,
immediately after BDL, were fed control diet or 1% taurocholate for 1 wk and rats that, immediately after BDL, underwent vagotomy and
subsequently were fed control diet or 1% taurocholate diet for 1 wk
was assessed by the 3 -hydroxysteroid dehydrogenase procedure by
absorption spectrophotometry using a commercially available kit (Wako
Chemicals, Richmond, VA) (34). Total bile acid
concentration was measured in the bile that was collected within the
first 10 min of bile flow. Aliquots of bile were analyzed for
individual bile salt by reversed-phase HPLC (13).
Purification of cholangiocytes.
Pure cholangiocytes were obtained from the selected group of animals by
immunoaffinity purification (4, 6, 7, 10, 17, 18, 30, 32).
Purity of cholangiocytes was evaluated by histochemistry for -GT
(46), a specific marker for cholangiocytes (8). Cell viability was >97%.
Evaluation of apoptosis. We evaluated cholangiocyte apoptosis by TUNEL analysis (32) in liver sections (n = 3) and by both annexin-V staining (28, 32) and the cell death-detection ELISA assay in pure cholangiocytes from rats that, immediately after BDL, were fed control diet or 1% taurocholate for 1 wk and rats that, immediately after BDL, underwent vagotomy and subsequently were fed control diet or 1% taurocholate diet for 1 wk. To evaluate whether vagotomy-induced apoptosis is mediated through the extrinsic or intrinsic pathway, we measured apoptosis by TUNEL analysis in liver sections and by both annexin-V staining and cell death-detection ELISA assay in purified cholangiocytes from BDL, vagotomized rats that were fed taurocholate and simultaneously treated by intraperitoneal injections with Z-IETD-fmk for 1 wk. Similarly, to evaluate whether the protective effect of taurocholate on vagotomy-induced apoptosis is mediated by PI3-kinase or MEK pathways, we measured apoptosis by TUNEL analysis in liver sections and by both annexin-V staining and cell death-detection ELISA assay in purified cholangiocytes from rats that, immediately after BDL + vagotomy, were fed 1% taurocholate and simultaneously treated by intraperitoneal injections with wortmannin (a PI3-kinase inhibitor) or U-0126 (an MEK inhibitor), respectively, for 1 wk.
TUNEL analysis was performed using a commercially available kit (Wako Chemicals, Tokyo, Japan). After liver sections were counterstained with hematoxylin solution, they were examined by light microscopy with an Olympus BX-40 microscope (Olympus Optical) equipped with a camera. Approximately 100 cells per slide were counted in a coded fashion in 10 nonoverlapping fields. After annexin-V staining (28, 32), cells were counterstained with hematoxylin solution and examined by light microscopy with an Olympus BX-40 microscope equipped with a camera. Approximately 300 cells per slide were counted in a coded fashion. Cell death detection by the Elisa assay was performed according to the vendor's instructions (Boehringer-Mannheim, Frankfurt, Germany). The cell death ELISA assay is an assay that detects apoptosis. It is based on the binding of the anti-histone antibody with the core histones H2A, H2B, H3, and H4 that form the mono/oligonucleosomes, typical of the apoptotic degeneration of the nuclei. Briefly, 1 × 105 pure cholangiocytes from the selected group of animals were resuspended in 500 µl of incubation buffer and incubated at 4°C for 30 min. Cells were then centrifuged, and the supernatant was rediluted in incubation buffer. The samples were incubated for 90 min at room temperature in microtiter plate (MTP) modules coated with a buffer containing an anti-histone antibody. The anti-histone antibody binds to the core histones H2A, H2B, H3, and H4 that form the mono/oligonucleosomes, typical of the apoptotic degeneration of the nuclei. After being washed, a solution containing anti-DNA-peroxidase was added and another incubation of 90 min followed. After removal of unbound peroxidase conjugate by washing, the amount of peroxidase retained in the immunocomplex was determined photometrically with 2,2'-azino-di[3-ethylbenzthiazoline sulfonate (6)] as a substrate. To evaluate whether taurocholate protects from vagotomy-induced apoptosis through changes in the caspase cascade by a PI3-kinase-dependent mechanism, we measured the activity of caspase 3, 8, and 9 in pure cholangiocytes from rats that, immediately after BDL, were fed control or 1% taurocholate diet for 1 wk, rats that, immediately after BDL, underwent vagotomy and were subsequently fed control or 1% taurocholate diet, and rats that, immediately after BDL + vagotomy, were fed 1% taurocholate and simultaneously treated by intraperitoneal injections with wortmannin (a PI3-kinase inhibitor) for 1 wk. Intracellular cholangiocyte caspase 3, 8, and 9 activities were measured by enzymatic kits according to the instructions supplied by the vendor (Medical & Biological Laboratories, Nagoya, Japan). The activity of caspases 3, 8, and 9 was measured as follows. Pure cholangiocytes, from the selected group of animals, were centrifuged at 1,500 rpm for 10 min, incubated in lysis buffer on ice for 10 min, and subsequently centrifuged at 10,000 g for 10 min. After centrifugation, the supernatant, containing the cytosolic fraction, was transferred to a clean tube. For each sample, 100 µg of proteins or BSA (negative control) were added to 50 µl of 2× reaction buffer. The activities of caspase 3, 8, and 9 were measured by proteolytic cleavage of substrates such as DEVD-pNA (caspase 3 substrate), IETD-pNA (caspase 8 substrate), and LEHD-pNA (caspase 9 substrate), respectively, added to each sample. The assay is based on the photometric detection of the chromophore p-nitoranilide (pNA) after cleavage from the substrates. The pNA light emission was quantified using a microtiter plate reader at 406 nm.Evaluation of cholangiocyte proliferation. Cholangiocyte proliferation was evaluated by quantitative immunohistochemistry for PCNA (32) or cytokeratin-19 (30) [CK-19, a cholangiocyte-specific marker (30)] in liver sections and by measurement of PCNA protein expression by immunoblot (17) in pure cholangiocytes from rats that, immediately after BDL, were fed control diet or 1% taurocholate for 1 wk, rats that, immediately after BDL, underwent vagotomy and subsequently were fed control diet or 1% taurocholate diet for 1 wk, and rats that, immediately after BDL + vagotomy, were fed 1% taurocholate and subsequently treated by intraperitoneal injections with wortmannin for 1 wk. After sections were stained for PCNA or CK-19, they were counterstained with hematoxylin and examined with a microscope (Olympus BX 40, Olympus Optical). Data were expressed as number of PCNA- or CK-19-positive cholangiocytes per each 100 cholangiocytes counted in seven different fields. DNA replication was evaluated by measurement of PCNA protein expression by immunoblot (17) in pure cholangiocytes from the aforementioned group of animals. The intensity of the bands was determined by scanning video densitometry using the ChemiImager 4000 low-light imaging system (Alpha Innotech, San Leandro, CA).
Measurement of basal and secretin-stimulated ductal secretion. Ductal secretion was evaluated by measurement of basal and secretin-stimulated bicarbonate-rich choleresis, secretin receptor (SR) gene expression, and basal and secretin-induced cAMP levels in pure cholangiocytes from rats that, immediately after BDL, were fed control diet or 1% taurocholate for 1 wk and rats that, immediately after BDL, underwent vagotomy and subsequently were fed control diet or 1% taurocholate diet for 1 wk.
After anesthesia, rats were surgically prepared for bile collection as described (8). One jugular vein was incannulated with a PE-50 cannula (Clay-Adams, New York, NY) to infuse either Krebs-Ringer-Henseleit (KRH) buffer or secretin (100 nM) dissolved in KRH. The rate of fluid infusion was adjusted according to both the rate of bile flow and the value of the arterial hematocrit and ranged from 0.738 to 2.964 ml/h. Body temperature was monitored with a rectal thermometer (Yellow Springs Instruments, Yellow Springs, OH) and maintained at 37°C by using a heating pad. As soon as the bile duct was incannulated (a step that takes only a few seconds because we need only to connect the plastic tubing coming out from the bile duct with another tube of bigger diameter) (8), collection of bile started immediately. When steady spontaneous bile flow was reached (60-70 min from the infusion of KRH), rats were infused for 30 min with secretin followed by a final infusion of KRH for 30 min. After the rats were surgically prepared for bile-flow experiments, bile was collected every 10 min in preweighed tubes and immediately stored atMeasurement of Akt phosphorylation.
To determine that the PI3-kinase-Akt pathway regulates taurocholate
prevention of vagotomy-induced duct damage, we evaluated, by
immunoblots (17) (in whole cell lysate from purified
cholangiocytes from rats that, immediately after BDL, were fed control
diet or 1% taurocholate for 1 wk and rats that, immediately after BDL, underwent vagotomy and subsequently were fed control diet or 1% taurocholate for 1 wk), the expression of the total protein and the
phosphorylation (Ser473) of Akt (a downstream effector of
the PI3-kinase-dependent cell-survival pathway responsible for
antiapoptotic activity) (42). To determine whether taurocholate changes in Akt phosphorylation are PI3-kinase dependent, we evaluated total and phosphorylated Akt in cholangiocytes from rats that, immediately after BDL, underwent vagotomy, were fed
taurocholate, and were subsequently treated with wortmannin [1 daily
intraperitoneal injection of 0.7 mg · kg1 · body wt
1
(38)] for 1 wk.
Role of ABAT on taurocholate prevention of vagotomy-induced effects on cholangiocyte apoptosis, proliferation, secretion, and Akt phosphorylation. To determine whether changes in cholangiocyte apoptosis, proliferation, secretion, and Akt phosphorylation are dependent on ABAT activity, we evaluated ABAT protein expression (5, 10) and bile acid transport activity (5, 10) in pure cholangiocytes from rats that, immediately after BDL, were fed control diet and rats that, immediately after BDL, underwent vagotomy and subsequently were fed control diet or 1% taurocholate diet for 1 wk. Because we have previously shown (10) that taurocholate feeding increases ABAT protein expression and bile acid transport activity in rat cholangiocytes, we did not evaluate ABAT protein expression and bile acid transport activity in cholangiocytes from BDL rats fed taurocholate for 1 wk.
To determine whether the taurocholate-induced changes in ABAT expression are PI3-kinase dependent, we evaluated ABAT protein expression in cholangiocytes from rats that, immediately after BDL + vagotomy, were fed taurocholate and were subsequently treated with wortmannin [1 daily intraperitoneal injection of 0.7 mg · kgStatistical analysis. All data are expressed as means ± SE. The differences between groups were analyzed by Student's t-test when two groups were analyzed or ANOVA if more than two were analyzed.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Assessment of biliary bile acid concentration and composition.
Bile acid composition of bile from the selected groups of rats is shown
in Table 1. After vagotomy, there was a
significant decrease in biliary bile acid concentration of muricholic,
taurocholic, and taurochenodeoxycholic acid compared with BDI control
rats (Table 1). Taurocholate feeding to BDI + vagotomy rats
increased total biliary bile acid concentration to levels higher than
those of BDI control rats (Table 1). Biliary bile acid concentration and composition of rats that, immediately after BDI, were fed 1%
taurocholate for 1 wk are also shown in Table 1.
|
Measurement of apoptosis.
taurocholate prevention of vagotomy-induced cholangiocyte
apoptosis is blocked by wortmannin.
In agreement with previous studies (33), TUNEL analysis
showed only a few apoptotic bodies in the liver sections of BDL rats fed control diet or 1% taurocholate for 1 wk (Fig.
1A). The number of
cholangiocytes undergoing apoptosis increased in liver sections
from BDL with vagotomy compared with BDL control rats (Fig.
1A). Taurocholate feeding prevented the increase in
cholangiocyte apoptosis induced by vagotomy (Fig.
1A).
|
|
|
|
|
Changes in cholangiocyte apoptosis, proliferation,
secretion, and Akt phosphorylation are dependent on ABAT activity.
We evaluated ABAT protein expression and bile acid transport activity
in pure cholangiocytes from BDL rats fed control diet and BDL + vagotomy rats fed control diet or taurocholate for 1 wk. In agreement
with previous studies (5), ABAT (with a band migrating at
~42 kDa) was expressed by cholangiocytes from BDL rats fed control
diet (Fig. 5A). Vagotomy
induced a marked decrease in ABAT protein expression in purified
cholangiocytes compared with cholangiocytes from BDL control rats (Fig.
5A). Feeding taurocholate to BDL, vagotomized rats prevented
the inhibitory effect of vagotomy on ABAT protein expression, which was
not statistically different from that of 1-wk-old BDL rats (Fig.
5A). Consistent with the concept that taurocholate effects
on cholangiocyte functions are regulated by the PI3-kinase system, the
in vivo administration of wortmannin to BDL, vagotomized rats blocked
the protective effects of taurocholate against vagotomy inhibition of
cholangiocyte ABAT protein expression (Fig. 5A).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The study demonstrates that, in the BDL rat, taurocholate feeding 1) prevents cholangiocyte apoptosis due to vagotomy (28); 2) prevents vagotomy-induced inhibition of cholangiocyte proliferation and secretin-stimulated ductal secretion (28); 3) prevents vagotomy-induced inhibition of PI3-kinase and activation of caspase 8, 9, and 3 activities; and 4) prevents vagotomy-induced inhibition of ABAT protein expression and bile acid transport activity. Taurocholate prevention of vagotomy-induced apoptosis was regulated by the PI3-kinase but not by the MEK pathway, because wortmannin (but not U-0126) blocked taurocholate protective effects against vagotomy-induced cholangiocyte apoptosis. Consistent with the concept that vagotomy-induced apoptosis is mediated by the intrinsic rather than the extrinsic pathway, the caspase 8 inhibitor Z-IETD-fmk did not alter vagotomy-induced increase in cholangiocyte apoptosis. Taurocholate prevention of vagotomy inhibition of cholangiocyte proliferation was regulated by the PI3-kinase, since wortmannin blocked-taurocholate protective effects against vagotomy inhibition of cholangiocyte proliferation. The modulatory effect of bile salts on cholangiocyte proliferation and secretion correlates with the protein expression and functional activity of ABAT (3, 7, 10), the transporter localized in the apical pole of cholangiocytes, and is responsible for bile salt entrance into cholangiocytes (5, 27).
We measured biliary bile acid concentration and composition and showed that vagotomy effects on cholangiocyte functions and taurocholate prevention of vagotomy effects on cholangiocyte functions are associated with altered bile acid composition. We found that after vagotomy, the biliary concentration of taurocholate decreases compared with BDL rats, whereas feeding taurocholate to BDL + vagotomy rats increased biliary concentration of taurocholate.
Intracellular accumulation of bile acids may induce apoptosis and liver injury (40). Not all the bile acids, however, are toxic. For example, some bile acids protect hepatocytes (40, 45, 49) from apoptosis. Taurocholate prevents the CCl4-induced apoptosis of cholangiocytes through phosphorylation of Akt (33). Bile acids accumulate in bile during cholestasis, thus representing a possible trigger for cholangiocyte proliferation (8, 10). In support of this concept, previous studies have shown that bile acids directly stimulate ductal proliferation (4, 7, 10). The current study extends this concept by showing that taurocholate is also able to sustain proliferation in a condition characterized by bile duct loss because of the lack of cholinergic innervation. Together, the reduced apoptosis and the increased proliferation, due to taurocholate feeding, maintained bile duct mass at levels not statistically different from those of the BDL rat (18). The importance of this protective effect is also demonstrated by our finding that, after taurocholate feeding, cholangiocytes maintain their functional activity, i.e., the enhanced ductal secretory response to secretin, SR gene expression, and increased basal and secretin-stimulated intracellular cAMP levels, which are lost in the vagotomized rats (28).
Because bile acids exert their functions in hepatocytes through the activation of the PI3-kinase (35, 45, 52), we evaluated the role of its transduction pathway in taurocholate prevention of vagotomy-induced damage of cholangiocytes. We found that the simultaneous administration of the PI3-kinase inhibitor wortmannin blocked the protective effect of taurocholate on the vagotomy-induced apoptosis and inhibition of proliferation. Consistently, we also observed that vagotomy resulted in diminished phosphorylation of Akt [an immediate downstream of PI3-kinase (21, 42)], which was prevented by taurocholate feeding. The simultaneous administration of wortmannin, however, abolished the increase in Akt phosphorylation observed after taurocholate feeding. Therefore, our study provides evidence that the activation of the PI3-kinase/Akt pathway triggers, similar to other cells (19, 26, 37), an antiapoptotic and proproliferative signaling in cholangiocytes.
A number of studies has shown that changes in the PI3-kinase/Akt
cascade modulate the activity of caspases (20, 45). For instance, the monocyte survival factor stimulation of PI3-kinase/Akt pathway reduces caspase 8, 9, and 3 activation (20, 48).
Renal tubular cell injury by cisplatinum is associated with an enhanced phosphorylation of Akt, inhibition of which enhances activation of
caspase 3 and caspase 9 (25). In accordance with these
previous studies, we show that vagotomy reduces PI3-kinase/Akt and
enhances the caspase activity. In addition, we demonstrated that the
vagotomy-induced apoptosis of cholangiocytes likely occurs
through the mitochondrial or intrinsic pathway (47, 54),
because we did not observe any changes in the number of apoptotic
cholangiocytes after vagotomy in the presence of the caspase 8 inhibitor Z-IETD-fmk. The extrinsic pathway, in fact, has an essential
upstream event: the activation of caspase 8 after ligands bind to death
receptors (54). Thus the absence of changes in
apoptosis after the inhibition of caspase 8 suggests that the
vagotomy-induced apoptosis is likely to happen through the
intrinsic rather the extrinsic pathway of apoptosis. Moreover,
the intrinsic pathway is involved in the induction of apoptosis
by stress signals, among which hormone or growth factor withdrawal are
considered (47, 54). Therefore, the finding that the
vagotomy-induced apoptosis is mediated through the intrinsic pathway is in accordance with the concept that the deprivation of
cholinergic innervation represents the loss of an important element for
the growth of the biliary tree (28). The intrinsic pathway
leads to apoptosis through the release of the cytochrome c from the mitochondria and the subsequent activation of
caspase 9 and caspase 3 but not caspase 8 (47, 54). In our
study, in fact, caspase 9 and caspase 3 were found significantly
increased in cholangiocytes after the vagotomy of the BDL rat, compared with cholangiocytes from BDL rats treated with control or 1%
taurocholate diet. Surprisingly, in these cells, an upregulated
activity of caspase 8 was also observed. If our data do not provide any
direct explanation for this phenomenon, they suggest that the
vagotomy-induced triggering of the intrinsic pathway of
apoptosis may directly or indirectly activate caspase 8 in the
same fashion as what has been previously shown in other cells
(47). Taurocholate feeding prevented the increase of the
caspase activity due to vagotomy, an effect that was abolished by the
simultaneous administration of the PI3-kinase inhibitor wortmannin.
These data suggest that taurocholate, by sustaining the activation of
the PI3-kinase/Akt cascade, reduces the activation of caspases, thus
preventing the vagotomy-induced apoptosis. Similar findings
have been described in hepatocytes (40, 45), in which
taurochenodeoxycholate prevents apoptosis through the
activation of PI3-kinase and the consequent inhibition of the caspase
activity (45, 49). In hepatocytes, however, the protective
effect of PI3-kinase occurs without activation of Akt but requires
recruitment of one of the atypical PKC isoforms, PKC
(45). The discrepancy between this (45) and
our present results is likely due to different mechanisms for
regulation of apoptosis in cholangiocytes and hepatocytes.
Recently, it has been shown that the antiapoptotic message of the
bile acid-activated PI3-kinase is mediated, at least in part, by the
MAP-kinase cascade (43, 44). Therefore, we evaluated the
role of the MAP-kinase cascade in the taurocholate protection of
vagotomy-induced bile duct damage, treating BDL, vagotomized and
taurocholate-fed animals with the MEK inhibitor U-0126. However, the
presence of U-0126 did not diminish the protective effect of
taurocholate on the vagotomy-induced apoptosis. The MAP-kinase
activation by bile acids is secondary to the interaction of the bile
acid itself with the epidermal growth factor (EGF) receptor (43,
44). Thus the fact that the effects of taurocholate seem not to
depend on the MAP-kinase cascade might be ascribed to the lack of
interaction of this bile acid with the EGF receptor in cholangiocytes.
Further studies to elucidate the role of the links among bile acids,
EGF receptor, and the MAP kinases in cholangiocytes are necessary.
To clarify the intracellular mechanisms by which taurocholate prevented the effects of vagotomy on cholangiocyte apoptosis, proliferation, and secretion, we studied the changes in ABAT protein expression and cholangiocyte bile acid transport activity. The rationale for these studies is based on the fact that bile acids, to exert their effects on cholangiocytes, must be internalized through ABAT (5, 27). Thus we anticipated that the effects of bile acids on cholangiocyte proliferation are determined by ABAT expression and bile acid transport activity in cholangiocytes. First, we observed, for the first time, that the bile acid transport in the biliary epithelium is also controlled by cholinergic innervation, because both the expression and the activity of ABAT were reduced after vagotomy. The impaired ABAT protein expression and activity in cholangiocytes could be the consequence of reduced biliary bile salt concentration caused by vagotomy. Chronic taurocholate feeding, in contrast, prevents the loss of ABAT. In a fashion similar to what has been previously shown in the ileum (22), the apical uptake of bile acids by cholangiocytes increased in proportion to bile acid lumenal concentration through direct regulation of ABAT gene and protein expression (10). Consistent with the concept that taurocholate effects are PI3-kinase dependent, the restoration of ABAT protein expression by this bile acid was abolished in the presence of wortmannin. For further confirmation of the key role played by ABAT, we have also shown that in vitro exposure of cholangiocytes to taurocholate strongly increased proliferation in cells expressing higher levels of ABAT (i.e., cholangiocytes from BDL rats and BDL, vagotomized, taurocholate-fed rats) compared with cholangiocytes from vagotomized rats, which express low levels of ABAT. This effect also was PI3-kinase dependent, because the preincubation with wortmannin prevented the increase of cholangiocyte proliferation induced by taurocholate. Other important evidence of the link among ABAT, taurocholate, and PI3-kinase is that the in vitro exposure to taurocholate induced a significant increase of Akt phosphorylation in cholangiocytes with a higher expression and activity of ABAT (e.g., purified from BDL rats or rats subjected to BDL, vagotomy, and taurocholate feeding) but not in cholangiocytes with low ABAT expression and activity (e.g., purified from rats subjected to BDL and vagotomy). The changes in Akt phosphorylation after the in vitro exposure to taurocholate were PI3-kinase dependent, because they were abolished by the preincubation with wormannin. Hence, the loss of ABAT expression and activity observed after vagotomy might determine the lack of cytoprotective effects by bile acids. When ABAT expression is restored, the presence of high concentrations of cytoprotective bile acids (taurocholate in the current study) prevents vagotomy-induced cholangiocyte damage. The novelty of these data is based on the demonstration of the existence of a dual relationship between bile acids and their apical transporter. On one side, we have the modulation (PI3-kinase mediated) of ABAT expression and activity by bile acids; on the other, we have the dependency of bile acid effects on ABAT expression. These findings candidate ABAT as a possible major regulator of cholangiocyte proliferation/loss in cholestatic liver diseases in response to injury/toxins. In support of this concept, we have shown (3) that depletion of endogenous bile acids, by prolonged external bile drainage, leads to downregulation of ABAT and decreased cholangiocyte proliferation and secretin-stimulated ductal secretion, whereas feeding taurocholate to normal rats increases ABAT expression and bile acid transport activity that leads to enhanced cholangiocyte proliferation and secretin-stimulated ductal secretion (10).
Our findings also indicate that taurocholate and cholinergic innervation cooperatively modulate the PI3-kinase/Akt survival pathway by regulating cholangiocyte intracellular cAMP levels. We have previously shown (28) that maintenance of cAMP levels by forskolin administration counteracts the effects of vagotomy on cholangiocyte apoptosis, proliferation, and secretion. In the current study, taurocholate feeding determined a restoration of both basal and secretin-stimulated cAMP synthesis (at levels not statistically different from those of cholangiocytes from BDL rats), thus preventing the effects of vagotomy. In our studies, intracellular cAMP levels change in parallel with PI3-kinase/Akt activity. In fact, after vagotomy, decreased cAMP levels were coupled with diminished activation of the PI3-kinase/Akt pathway, followed by increased caspase activity, increased apoptosis, and diminished proliferation. In contrast, when vagotomized rats were fed with taurocholate, increased cAMP levels were associated with enhanced PI3-kinase/Akt activation, diminished caspase activity and apoptosis, and enhanced proliferation. The fundamental role of cAMP in mediating the effects of taurocholate is also demonstrated by the finding that the increase of cholangiocyte proliferation induced by the in vitro exposure to taurocholate is abolished when PKA [known to be an immediate downstream of cAMP (12, 16)] is inhibited by Rp-cAMP. In addition, the preincubation with Rp-cAMP also blocked the taurocholate-induced increase of PI3-kinase activity, as testified by the lack of the enhancement of Akt phosphorylation observed after the in vitro exposure to taurocholate. Our data are in accordance with previous studies in hepatocytes (52, 53), in which cAMP has been found to exert prosurvival effects through the modulation of the PI3-kinase-signaling pathway. Therefore, the modulation of cAMP synthesis appears to be the intracellular step at which the cholinergic and taurocholate pathways may merge.
The proposed intracellular pathway that allows taurocholate to exert
its protective effect against vagotomy-induced bile duct damage is
shown in Fig. 8. Taurocholate is
internalized in cholangiocytes through ABAT; once inside the cell,
taurocholate enhances the cAMP synthesis, which is responsible for the
increased PI3-kinase activity. PI3-kinase stimulates proliferation and
inhibits the caspase activity, thus leading to diminished
apoptosis. Taurocholate/cAMP-stimulated PI3-kinase is also
responsible for changes in the expression of the ABAT protein, which
represents the last step of a circle of events that are positively
regulated by the chronic exposure to high levels of taurocholate.
|
Previous studies (1, 10, 11) have observed how both bile acids and acetylcholine are able to modulate the intracellular Ca2+ release in cholangiocytes. Thus the prevention of vagotomy effects by taurocholate could involve also the Ca2+/Ca2+-dependent PKC signaling, likely as an upstream regulator of the cAMP synthesis.
These findings have important implications for the pathophysiology of the transplanted (denervated) liver (8), in which ischemic (39) or infectious (23) insults against intrahepatic bile ducts may not be adequately counteracted, during the immediate posttransplant period. With an inadequate repair mechanism due to the lack of cholinergic modulation of cholangiocyte proliferation, bile duct loss ensues (28). Supporting this hypothesis, biliary complications after liver transplant occur most often during the first 3 mo while reinnervation of the transplanted liver is occurring.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by a grant from MURST 40% (MM06215421/2) progetto nazionale 2000 (to D. Alvaro), by a grant award from Scott & White Hospital and Texas A&M University (to G. Alpini and G. D. LeSage), by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54208 (to G. D. LeSage) and DK-58411, by a Veterans Affairs merit award (to G. Alpini), by a grant-in-aid for Scientific Research (13670488) from Japan Society for the Promotion of Science (to Y. Ueno), by a grant award (MURSTMM06215421; to the Dept. of Gastroenterology, Univ. of Ancona, Ancona, Italy), and by The MURST Italian Grant 9806210866-006 (to L. Baiocchi).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: G. Alpini, The Texas A&M Univ. System, Health Sciences Center, College of Medicine, and Central Texas Veterans Health Care System, MRB, 702 South West H.K. Dodgen Loop, Temple, TX 76504 (E-mail: falpini{at}mailbox.sw.org).
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.
First published January 29, 2003;10.1152/ajpgi.00398.2002
Received 16 September 2002; accepted in final form 19 January 2003.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alpini, G,
Baiocchi L,
Glaser S,
Ueno Y,
Marzioni M,
Francis H,
Phinizy JL,
Angelico M,
and
LeSage G.
Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte proliferation and secretion of bile duct ligated rats through activation of PKC alpha.
Hepatology
35:
1041-1052,
2002[ISI][Medline].
2.
Alpini, G,
Benedetti A,
Marucci L,
Glaser S,
and
LeSage G.
Taurocholate (TC) but not taurolithocholate (TLC) abrogates carbon tetrachloride (CCl4)-induced cholangiocyte apoptosis by a phosphatidylinositol 3-kinase (PI3K)-dependent pathway.
Hepatology
30:
A897,
1999.
3.
Alpini, G,
Glaser S,
Alvaro D,
Ueno Y,
Marzioni M,
Francis H,
Baiocchi L,
Stati T,
Barbaro B,
Phinizy JL,
Mauldin J,
and
LeSage G.
Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase-dependent pathway in rats.
Gastroenterology
123:
1226-1237,
2002[ISI][Medline].
4.
Alpini, G,
Glaser S,
Robertson W,
Phinizy JL,
Rodgers RE,
Caligiuri A,
and
LeSage G.
Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes.
Am J Physiol Gastrointest Liver Physiol
273:
G518-G529,
1997
5.
Alpini, G,
Glaser SS,
Rodgers R,
Phinizy JL,
Robertson WE,
Lasater J,
Caligiuri A,
Tretjak Z,
and
LeSage GD.
Functional expression of the apical Na+-dependent bile acid transporter in large but not small rat cholangiocytes.
Gastroenterology
113:
1734-1740,
1997[ISI][Medline].
6.
Alpini, G,
Glaser SS,
Ueno Y,
Pham L,
Podila PV,
Caligiuri A,
LeSage G,
and
LaRusso NF.
Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation.
Am J Physiol Gastrointest Liver Physiol
274:
G767-G775,
1998
7.
Alpini, G,
Glaser SS,
Ueno Y,
Rodgers R,
Phinizy JL,
Francis H,
Baiocchi L,
Holcomb LA,
Caligiuri A,
and
LeSage GD.
Bile acid feeding induces cholangiocyte proliferation and secretion: evidence for bile acid-regulated ductal secretion.
Gastroenterology
116:
179-186,
1999[ISI][Medline].
8.
Alpini, G,
Lenzi R,
Sarkozi L,
and
Tavoloni N.
Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.
J Clin Invest
81:
569-578,
1988[ISI][Medline].
9.
Alpini, G,
Prall RT,
and
LaRusso NF.
The pathobiology of biliary epithelia.
In: The Liver; Biology and Pathobiology (4th ed.), edited by Arias IM,
Boyer JL,
Chisari FV,
Fausto N,
Jakoby W,
Schachter D,
and Shafritz DA.. Philadelphia: Lippincott, Williams & Wilkins, 2001, p. 421-435.
10.
Alpini, G,
Ueno Y,
Glaser SS,
Marzioni M,
Phinizy JL,
Francis H,
and
LeSage G.
Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes.
Hepatology
34:
868-876,
2001[ISI][Medline].
11.
Alvaro, D,
Alpini G,
Jezequel AM,
Bassotti C,
Francia C,
Fraioli F,
Romeo R,
Marucci L,
Le Sage G,
Glaser SS,
and
Benedetti A.
Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions.
J Clin Invest
100:
1349-1362,
1997
12.
Alvaro, D,
Mennone A,
and
Boyer JL.
Role of kinases and phosphatases in the regulation of fluid secretion and Cl/HCO
13.
Cantafora, A,
Di Biase A,
Alvaro D,
and
Angelico M.
An improved method for measuring the glycine and taurine conjugates of bile salts by high performance liquid chromatography with tauro-7-,12-
-dihydroxy-5-
-cholanic acid is internal standard.
J Chromatogr
386:
367-370,
1987[Medline].
14.
Clemons, AP,
Holstein DM,
Galli A,
and
Saunders C.
Cerulein-induced acute pancreatitis in the rat is significantly ameliorated by treatment with MEK1/2 inhibitors U0126 and PD98059.
Pancreas
25:
251-259,
2002[ISI][Medline].
15.
Ferrer, I.
Role of caspases in ionizing radiation-induced apoptosis in the developing cerebellum.
J Neurobiol
41:
549-558,
1999[ISI][Medline].
16.
Fouassier, L,
Duan CY,
Feranchak AP,
Yun CH,
Sutherland E,
Simon F,
Fitz JG,
and
Doctor RB.
Ezrin-radixin-moesin-binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia.
Hepatology
33:
166-176,
2001[ISI][Medline].
17.
Glaser, S,
Benedetti A,
Marucci L,
Alvaro D,
Baiocchi L,
Kanno N,
Caligiuri A,
Phinizy JL,
Chowdhury U,
Papa E,
LeSage G,
and
Alpini G.
Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C-dependent mechanisms.
Hepatology
32:
17-25,
2000[ISI][Medline].
18.
Glaser, SS,
Rodgers RE,
Phinizy JL,
Robertson WE,
Lasater J,
Caligiuri A,
Tretjak Z,
LeSage GD,
and
Alpini G.
Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes.
Am J Physiol Gastrointest Liver Physiol
273:
G1061-G1070,
1997
19.
Goncharova, EA,
Ammit AJ,
Irani C,
Carroll RG,
Eszterhas AJ,
Panettieri RA,
and
Krymskaya VP.
PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
283:
L354-L363,
2002
20.
Goyal, A,
Wang Y,
Graham MM,
Doseff AI,
Bhatt NY,
and
Marsh CB.
Monocyte survival factors induce Akt activation and suppress caspase-3.
Am J Respir Cell Mol Biol
26:
224-230,
2002
21.
Graf, D,
Kurz AK,
Fischer R,
Reinehr R,
and
Haussinger D.
Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N-terminal kinase-dependent manner.
Gastroenterology
122:
1411-1427,
2002[ISI][Medline].
22.
Hofmann, AF.
Regulation of ileal bile acid transport: desirability of measuring transport function as well as transporter activity.
Hepatology
29:
1335-1337,
1999[ISI][Medline].
23.
Humar, A,
Kumar D,
Caliendo AM,
Moussa G,
Ashi-Sulaiman A,
Levy G,
and
Mazzulli T.
Clinical impact of human herpesvirus 6 infection after liver transplantation.
Transplantation
73:
599-604,
2002[ISI][Medline].
24.
Ishii, M,
Vroman B,
and
LaRusso NF.
Isolation and morphological characterization of bile duct epithelial cells from normal rat liver.
Gastroenterology
97:
1236-1247,
1989[ISI][Medline].
25.
Kaushal, GP,
Kaushal V,
Hong X,
and
Shah SV.
Role and regulation of activation of caspases in cisplatin-induced injury to renal tubular epithelial cells.
Kidney Int
60:
1726-1736,
2001[ISI][Medline].
26.
Kennedy, SG,
Wagner AJ,
Conzen SD,
Jordan J,
Bellacosa A,
Tsichlis PN,
and
Hay N.
The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal.
Genes Dev
11:
701-713,
1997[Abstract].
27.
Lazaridis, KN,
Pham L,
Tietz P,
Marinelli RA,
deGroen PC,
Levine S,
Dawson PA,
and
LaRusso NF.
Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter.
J Clin Invest
100:
2714-2721,
1997
28.
LeSage, G,
Alvaro D,
Benedetti A,
Glaser S,
Marucci L,
Baiocchi L,
Eisel W,
Caligiuri A,
Phinizy JL,
Rodgers R,
Francis H,
and
Alpini G.
Cholinergic system modulates growth, apoptosis, and secretion of cholangiocytes from bile duct-ligated rats.
Gastroenterology
117:
191-199,
1999[ISI][Medline].
29.
LeSage, G,
Glaser S,
Ueno Y,
Alvaro D,
Baiocchi L,
Kanno N,
Phinizy JL,
Francis H,
and
Alpini G.
Regression of cholangiocyte proliferation after cessation of ANIT feeding is associated with increased apoptosis.
Am J Physiol Gastrointest Liver Physiol
281:
G182-G190,
2001
30.
LeSage, G,
Glaser SS,
Gubba S,
Robertson WE,
Phinizy JL,
Lasater J,
Rodgers RE,
and
Alpini G.
Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased secretin-induced ductal secretion.
Gastroenterology
111:
1633-1644,
1996[ISI][Medline].
31.
LeSage, GD,
Benedetti A,
Glaser S,
Marucci L,
Tretjak Z,
Caligiuri A,
Rodgers R,
Phinizy JL,
Baiocchi L,
Francis H,
Lasater J,
Ugili L,
and
Alpini G.
Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver.
Hepatology
29:
307-319,
1999[ISI][Medline].
32.
LeSage, GD,
Glaser SS,
Marucci L,
Benedetti A,
Phinizy JL,
Rodgers R,
Caligiuri A,
Papa E,
Tretjak Z,
Jezequel AM,
Holcomb LA,
and
Alpini G.
Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver.
Am J Physiol Gastrointest Liver Physiol
276:
G1289-G1301,
1999
33.
Marucci, L,
Alpini G,
Glaser S,
Alvaro D,
Benedetti A,
Francis H,
Phinizy JL,
Marzioni M,
Mauldin J,
Venter J,
Baumann B,
Ugili L,
and
LeSage G.
Taurocholate feeding prevents CCl4-induced damage of large cholangiocytes through a PI3 kinase dependent mechanism.
Am J Physiol Gastrointest Liver Physiol
284:
G290-G301,
2003
34.
Mashige, F,
Tanaka N,
Maki A,
Kamei S,
and
Yamanaka M.
Direct spectrophotometry of total bile acids in serum.
Clin Chem
27:
1352-1356,
1981
35.
Misra, S,
Ujhazy P,
Gatmaitan Z,
Varticovski L,
and
Arias IM.
The role of phosphoinositide 3-kinase in taurocholate-induced trafficking of ATP-dependent canalicular transporters in rat liver.
J Biol Chem
273:
26638-26644,
1998
36.
Namura, S,
Iihara K,
Takami S,
Nagata I,
Kikuchi H,
Matsushita K,
Moskowitz MA,
Bonventre JV,
and
Alessandrini A.
Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia.
Proc Natl Acad Sci USA
98:
11569-11574,
2001
37.
Neri, LM,
Borgatti P,
Capitani S,
and
Martelli AM.
The nuclear phosphoinositide 3-kinase/AKT pathway: a new second messenger system.
Biochim Biophys Acta
1584:
73,
2002[ISI][Medline].
38.
Ng, SS,
Tsao MS,
Nicklee T,
and
Hedley DW.
Wortmannin inhibits pkb/akt phosphorylation and promotes gemcitabine antitumor activity in orthotopic human pancreatic cancer xenografts in immunodeficient mice.
Clin Cancer Res
7:
3269-3275,
2001
39.
Noack, K,
Bronk SF,
Kato A,
and
Gores GJ.
The greater vulnerability of bile duct cells to reoxygenation injury than to anoxia. Implications for the pathogenesis of biliary strictures after liver transplantation.
Transplantation
56:
495-500,
1993[ISI][Medline].
40.
Patel, T,
Roberts LR,
Jones BA,
and
Gores GJ.
Dysregulation of apoptosis as a mechanism of liver disease: an overview.
Semin Liver Dis
18:
105-114,
1998[ISI][Medline].
41.
Powley, TL,
Prechtl JC,
Fox EA,
and
Berthoud HR.
Anatomical considerations for surgery of the rat abdominal vagus: distribution, paraganglia and regeneration.
J Auton Nerv Syst
9:
79-97,
1983[ISI][Medline].
42.
Pugazhenthi, S,
Nesterova A,
Sable C,
Heidenreich KA,
Boxer LM,
Heasley LE,
and
Reusch JE.
Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein.
J Biol Chem
275:
10761-10766,
2000
43.
Qiao, L,
Studer E,
Leach K,
McKinstry R,
Gupta S,
Decker R,
Kukreja R,
Valerie K,
Nagarkatti P,
El Deiry W,
Molkentin J,
Schmidt-Ullrich R,
Fisher PB,
Grant S,
Hylemon PB,
and
Dent P.
Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced apoptosis.
Mol Biol Cell
12:
2629-2645,
2001
44.
Qiao, L,
Yacoub A,
Studer E,
Gupta S,
Pei XY,
Grant S,
Hylemon PB,
and
Dent P.
Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes.
Hepatology
35:
779-789,
2002[ISI][Medline].
45.
Rust, C,
Karnitz LM,
Paya CV,
Moscat J,
Simari RD,
and
Gores GJ.
The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade.
J Biol Chem
275:
20210-20216,
2000
46.
Rutemburg, AM,
Kim H,
Fishbein JW,
Hanker JS,
Wasserkrug HL,
and
Seligman AM.
Histochemical and ultrastructural demonstration of -glutamyl transpeptidase activity.
J Histochem Cytochem
17:
517-526,
1969[ISI][Medline].
47.
Sitailo, LA,
Tibudan SS,
and
Denning MF.
Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes.
J Biol Chem
277:
19346-19352,
2002
48.
Suhara, T,
Kim HS,
Kirshenbaum LA,
and
Walsh K.
Suppression of Akt signaling induces Fas ligand expression: involvement of caspase and Jun kinase activation in Akt-mediated Fas ligand regulation.
Mol Cell Biol
22:
680-691,
2002
49.
Takikawa, Y,
Miyoshi H,
Rust C,
Roberts P,
Siegel R,
Mandal PK,
Millikan RE,
and
Gores GJ.
The bile acid-activated phosphatidylinositol 3-kinase pathway inhibits Fas apoptosis upstream of bid in rodent hepatocytes.
Gastroenterology
120:
1810-1817,
2001[ISI][Medline].
50.
Tietz, PS,
Alpini G,
Pham LD,
and
Larusso NF.
Somatostatin inhibits secretin-induced ductal hypercholeresis and exocytosis by cholangiocytes.
Am J Physiol Gastrointest Liver Physiol
269:
G110-G118,
1995
51.
Vaughn, WK,
Neal RA,
and
Anderson AJ.
Computer estimation of the parameters of sigmoid kinetic model.
Comput Biol Med
6:
1-7,
1976[Medline].
52.
Webster, CR,
and
Anwer MS.
Cyclic adenosine monophosphate-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes.
Hepatology
27:
1324-1331,
1998[ISI][Medline].
53.
Webster, CR,
Usechak P,
and
Anwer MS.
cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochrome c release.
Am J Physiol Gastrointest Liver Physiol
283:
G727-G738,
2002
54.
Yin, XM.
Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways.
Cell Res
10:
161-167,
2000[ISI][Medline].