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Bidirectional water transport across biliary epithelia
GASTROENTEROLOGY Vol. 118, No.4
A930 AASLD ABSTRACTS
149
151
CORTICOSTEROIDS MODULATE THE SECRETORY PRO· CESSES OF THE RAT INTRAHEPATIC BILIARY EPITHELIUM.
BILE ACID ABSORPTION IN ISOLATED BILE DUCT UNITS (IBDU) IS PRIMARILY DUE TO THE APICAL BILE ACID TRANSPORTER.
Domenico Alvaro, Luca Marucci, Alessandro Gigliozzi, Gianfranco Apini, Emanuela Papa, Marco Delle Monache, Rita Monterubbianesi, Livio Capocaccia, Antonio Benedetti, Univ of Rome La sapienza, Rome, Italy; Univ of Ancona, Ancona, Italy; Univ La Sapienza, Rome, Italy; Texas A&M Univ and Cent Texas Veterans HSC, Temple, TX. One of the major functions of cholangiocytes is to secrete bicarbonate ions in bile through the coordinate action of several H+ICO; transport processes including the apically located Cl-IHCOj exchanger. Although corticosteroids are currently under evaluation for the treatment of chelangiopathies, their effects on the functions of the intrahepatic biliary epithelium are unknown. We investigated both in vivo and in vitro the expression of glucocorticoid receptors (GcR) in the intrahepatic biliary epithelium and the role of corticosteroids in the regulation of cholangiocyte secretory processes. METHODS: The expression of GcR was studied in situ in liver sections from normal and bile duct ligated (BDL) rats by immunohistochemistry and in pure isolated rat cholangiocytes by immunohistochemistry and RT-PCR. The effects of dexamethasone and budesonide on bicarbonate biliary excretion and H+tHCO; transport processes were investigated in bile fistula rats and isolated intrahepatic bile duct units (IBDU). RESULTS: We found, by both immunohistochemistry and RT-PCR, that GcR were expressed in rat cholangiocytes and were markedly increased after cholangiocyte proliferation induced by BDL. Cholangiocytes expressed GcR at a higher level when compared with hepatocytes. Acute administration of dexamethasone and budesonide failed to influence bicarbonate biliary secretion in bile fistula rats and H+ IHC03- transport processes in IBDU. However, treatment for 2-3 days with dexamethasone (1 mg tid) or budesonide (0.3 mg tid) induced a significant (p<0.05) increase in bicarbonate biliary concentration and secretion. IBDU incubated for 24-36 hours with dexamethasone (I-50 /-LM) or budesonide (0.5-10 /-LM) showed a significant (p<0.02) increase in the expression (+ 120%, by western blot) and activity (+40-60%) of the Na+lH+ exchanger (NHE 1 isoform) and of the CI·tHCO; exchanger. CONCLUSIONS: The intrahepatic biliary epithelium expresses GcR, which are up-regulated during cholangiocyte proliferation. Stimulation with corticosteroids enhances the expression and activities of transport processes driving bicarbonate excretion in bile suggesting a potential mechanism for the beneficial effects of corticosteroids in the treatment of cholangiopathies. Supported by Grant # 9806210866 from MURST.
150 THE ALPHA-2 ADRENERGIC AGONIST, UK 14,304, INHIBITS GROWTH AND INCREASES APOPTOSIS OF THE CHOLANGIO· CARCINOMA CELL LINE, MZ·CHA·l, THROUGH CAMP BUT NOT CA2+·DEPENDENT MECHANISMS.
Gianfranco Alpini, Shannon Glaser, Jo Lynne Phinizy, Alan F. Hofmann, Gene LeSage, Texas A&M Univ and Cent Texas Veterans HSC, Temple, TX; Scott & White Hosp, Temple, TX; Scott & White, Temple, TX; Univ of CA, San Diego, CA. The Na+-dependent apical bile acid transporter (ABAT) is present in the apical domain of cholangiocytes. We have shown that elevated cholangiocyte cAMP levels increase ABAT activity in vitro. The physiologic relevance, transport characteristics and amount of bile acid absorption from bile by ABAT remains unclear. We propose the hypothesis that ABAT is the primary bile acid transporter in cholangiocytes. AIMS: To determine bile acid transport characteristics of bile ducts. Methods: Isolated IBDU were isolated from rat liver. The IBDU lumen was impaled with two micropipettes. Solutions were perfused into the IBDU lumen with one pipette and luminal contents were concurrently collected with the second pipette. IBDU were perfused with fluorescent bile acid analogs [Cholyl(Ne-NBD)-lysine, (C-L-NBD) (previously shown to have high affinity for ABAT in the ileum) or cholylamidofluorescein (CAMF) (which has low affinity for ABAT)] and Texas Red-dextran as a fluid phase recovery marker. The percentage of ductal bile acid absorption was determined from the ratio of collected to perfusate fluorescence. Bile acid fluorescence was expressed as a ratio to Texas Red fluorescence to correct for dilution from IBDU secretion and potential leakage of fluorescent bile acid from the impaled sites. Cholangiocyte cAMP levels were increased by the addition of forskolin (10.4 M). RESULTS: IBDU luminal diameter (l00-200 /-Lm) remained unchanged during the perfusion experiments. The percentage of perfused C-L-NBD and CAMF absorbed by IBDU was 13% and 2% respectively. 77% of C-L-NBD absorption was dependent on the presence of Na+ in the perfusate. Forskolin increased Na+-dependent C-L-NBD absorption to 22% (p<0.05 compared to control) but had no effect on CAMF absorption. The absence of bicarbonate or CI' did not alter basal or forskolin-stimulated C-L-NBD absorption. C-L-NBD fluorescence did not accumulate in the cytoplasm of cholangiocytes suggesting apical bile acid uptake is the rate limiting step. SUMMARY/CONCLUSIONS: We have established a microperfused system for the study of bile acid absorption in IBDU. Consistent with an ABAT transport mechanism, ductal absorption is primarily Na+-dependent, showed substrate specificity identical to ABAT in the ileum, is not dependent on an anion exchange mechanism, and increases with elevated cholangiocyte cAMP levels. These data are consistent with our hypothesis that ABAT is the primary bile acid transporter in cholangiocytes.
152
Noriatsu Kanno, Shannon Glaser, Usha Chowdhury, Jo Lynne Phinizy, Heather Francis, Gene LeSage, Gianfranco Alpini, Texas A&M Univ, Temple, TX; Scott & White Hosp, Temple, TX; Texas A&M Univ and Cent Texas Veterans HSC, Temple, TX.
BIDIRECTIONAL WATER TRANSPORT ACROSS BILIARY EPI· THELIA.
Cholangiocytes are the target cells during hyperplastic and neoplastic growth in the liver. Cholangiocarcinoma, derived from intrahepatic and extrahepatic bile duct epithelial cells, displays a poor prognosis without established measures for treatment. In addition, the regulation of its growth is not fully defined. We have shown that the parasympathetic system plays an important role in the regulation of hyperplastic cholangiocyte growth and apoptosis. Adrenergic receptors regulate hyperplastic and neoplastic growth in a number of cell types through cAMP or 1P3-dependent mechanisms. No information exists regarding the role of the adrenergic system in the regulation of cholangiocarcinoma growth. AIMS: To evaluate the role and mechanisms of action of alpha-2 adrenergic receptors in the modulation of cholangiocarcinoma growth. METHODS: Studies were performed in the human cholangiocarcinoma cell line, Mz-ChA-l (a gift from Dr. G. Fitz). We first studied: (i) the expression of three subtypes (alpha2A, alpha-2B and alpha-2C) of adrenergic receptors in Mz-ChA-I cells by both immunocytochemistry and Western blotting analysis; and (ii) the effect of the alpha-2 adrenergic agonist (UK 14,304, 10 ILM) on the intracellular levels of cAMP and IP]. We determined the effect of UK 14,304 on cholangiocarcinoma DNA synthesis (evaluated by measurement of 3H-thymidine incorporation and PCNA protein expression) and apoptosis (determined by annexin-V staining) in the presence or absence of BAPTNAM (an intracellular calcium chelator), H7 (a Ca2i- -dependent PKCainhibitor), or rottlerin (a Ca2+-independent PKC-Ilinhibitor). RESULTS: Mz-ChA-l cells expressed alpha-2A, alpha-2B and alpha-2C receptors. UK14,304 inhibited the growth of Mz-ChA-1 cells in a doseand time-dependent manner. The inhibitory effect of UK 14,304 on cholangiocarcinoma growth was not affected by BAPT N AM, H7 or rottlerin but was associated with decreased levels of cAMP but not by IP 3. This inhibitory effect of UK14,304 was associated with increased apoptosis of Mz-ChA-l cells. SUMMARY/CONCLUSIONS: Mz-ChA-l cells expressed functional alpha-2 adrenergic receptors. The antiproliferative effect of UK 14,304 on cholangiocarcinoma growth was associated with increased apoptosis through the cAMP system. The data suggest that modulation of the adrenergic system could be exploited in the management of cholangiocarcinoma growth.
Progress in understanding ductal bile formation requires techniques that allow measurement of transepithelial water transport in intact bile ducts. We previously reported an experimental approach that allows microperfusion of the lumen of intrahepatic bile duct units (IBDUs) isolated from normal rat liver (Gastroenterology l16:A1236, 1999). Our AIM here was to study transepithelial water transport in perfused IBDUs using an epifiuorescence technique to provide quantitative water transport data in real time. METHODS. IBDUs (100-150 /-Lm diameter, 1-1.5 mm length) isolated from normal rat liver were perfused in vitro with a membraneimpermeant f1uorophore, fluorescein-sulfonate (FS), as a volume marker. Microperfused IBDUs were epiilluminated to excite FS fluorescence in a small spot (50-100 /-Lm) at the distal end of the lumen of IBDUs. Net water movement (Jv) and osmotic water permeability (Pf) were calculated from the FS concentration, perfusate rate flow, lumen and bath osmolalities, length of the IBDU, and surface area of the lumen. RESULTS. In the absence of an osmotic gradient (i.e., equal perfusate and bath buffer osmolality at 290 mOsm), the FS fluorescence was proportional to FS concentration, independent of perfusion flow rate, and constant along the length of perfused IBDUs. In contrast, when an inward osmotic gradient was established across IBDUs (perfusate osmolality-290 mOsm; bath osmolality-70 mOsm), the FS fluorescence at the distal end of the IBDU promptly (~30 sec) decr:ase~ indicati~§ water ~ov:ment ~om bath .to lumen, i.e., secretion (Pf-4.8_0.8 x 10 ern/sec; Jv--39.l_6.1 nl/mm/ mm). When a net outward osmotic gradient was established across IBDUs (perfusate osmolality-290 mOsm; bath osmolality-530 mOsm), the FS fluorescence promptly (~30 sec) increased, indicating water movement from lumen to bath, i.e., absorption (Pf=1.0:'::0.1 x 10_.3 ern/sec, Jv=24.l :'::2.7 nl/min/mm). Bath reperfusion of IBDUs with isotonic KRB returned FS fluorescence to its original level. CONCLUSION. Based on experimental models of water transport across other epithelial barriers, we developed a new experimental approach to study rapid changes in transepithelial water transport in intact bile ducts. The results indicate a relatively high Jv and Pf for IBDUs, providing functional evidence that intrahepatic bile ducts can both secrete and absorb water in response to osmotic gradients, presumably through molecular water channels.
Anatoly I. Masyuk, Michael J. Burke, Ai-Yu Gong, Nicholas F. LaRusso, Mayo Med Sch, Clin and Fdn, Rochester, MN.
A930 AASLD ABSTRACTS
149
151
CORTICOSTEROIDS MODULATE THE SECRETORY PRO· CESSES OF THE RAT INTRAHEPATIC BILIARY EPITHELIUM.
BILE ACID ABSORPTION IN ISOLATED BILE DUCT UNITS (IBDU) IS PRIMARILY DUE TO THE APICAL BILE ACID TRANSPORTER.
Domenico Alvaro, Luca Marucci, Alessandro Gigliozzi, Gianfranco Apini, Emanuela Papa, Marco Delle Monache, Rita Monterubbianesi, Livio Capocaccia, Antonio Benedetti, Univ of Rome La sapienza, Rome, Italy; Univ of Ancona, Ancona, Italy; Univ La Sapienza, Rome, Italy; Texas A&M Univ and Cent Texas Veterans HSC, Temple, TX. One of the major functions of cholangiocytes is to secrete bicarbonate ions in bile through the coordinate action of several H+ICO; transport processes including the apically located Cl-IHCOj exchanger. Although corticosteroids are currently under evaluation for the treatment of chelangiopathies, their effects on the functions of the intrahepatic biliary epithelium are unknown. We investigated both in vivo and in vitro the expression of glucocorticoid receptors (GcR) in the intrahepatic biliary epithelium and the role of corticosteroids in the regulation of cholangiocyte secretory processes. METHODS: The expression of GcR was studied in situ in liver sections from normal and bile duct ligated (BDL) rats by immunohistochemistry and in pure isolated rat cholangiocytes by immunohistochemistry and RT-PCR. The effects of dexamethasone and budesonide on bicarbonate biliary excretion and H+tHCO; transport processes were investigated in bile fistula rats and isolated intrahepatic bile duct units (IBDU). RESULTS: We found, by both immunohistochemistry and RT-PCR, that GcR were expressed in rat cholangiocytes and were markedly increased after cholangiocyte proliferation induced by BDL. Cholangiocytes expressed GcR at a higher level when compared with hepatocytes. Acute administration of dexamethasone and budesonide failed to influence bicarbonate biliary secretion in bile fistula rats and H+ IHC03- transport processes in IBDU. However, treatment for 2-3 days with dexamethasone (1 mg tid) or budesonide (0.3 mg tid) induced a significant (p<0.05) increase in bicarbonate biliary concentration and secretion. IBDU incubated for 24-36 hours with dexamethasone (I-50 /-LM) or budesonide (0.5-10 /-LM) showed a significant (p<0.02) increase in the expression (+ 120%, by western blot) and activity (+40-60%) of the Na+lH+ exchanger (NHE 1 isoform) and of the CI·tHCO; exchanger. CONCLUSIONS: The intrahepatic biliary epithelium expresses GcR, which are up-regulated during cholangiocyte proliferation. Stimulation with corticosteroids enhances the expression and activities of transport processes driving bicarbonate excretion in bile suggesting a potential mechanism for the beneficial effects of corticosteroids in the treatment of cholangiopathies. Supported by Grant # 9806210866 from MURST.
150 THE ALPHA-2 ADRENERGIC AGONIST, UK 14,304, INHIBITS GROWTH AND INCREASES APOPTOSIS OF THE CHOLANGIO· CARCINOMA CELL LINE, MZ·CHA·l, THROUGH CAMP BUT NOT CA2+·DEPENDENT MECHANISMS.
Gianfranco Alpini, Shannon Glaser, Jo Lynne Phinizy, Alan F. Hofmann, Gene LeSage, Texas A&M Univ and Cent Texas Veterans HSC, Temple, TX; Scott & White Hosp, Temple, TX; Scott & White, Temple, TX; Univ of CA, San Diego, CA. The Na+-dependent apical bile acid transporter (ABAT) is present in the apical domain of cholangiocytes. We have shown that elevated cholangiocyte cAMP levels increase ABAT activity in vitro. The physiologic relevance, transport characteristics and amount of bile acid absorption from bile by ABAT remains unclear. We propose the hypothesis that ABAT is the primary bile acid transporter in cholangiocytes. AIMS: To determine bile acid transport characteristics of bile ducts. Methods: Isolated IBDU were isolated from rat liver. The IBDU lumen was impaled with two micropipettes. Solutions were perfused into the IBDU lumen with one pipette and luminal contents were concurrently collected with the second pipette. IBDU were perfused with fluorescent bile acid analogs [Cholyl(Ne-NBD)-lysine, (C-L-NBD) (previously shown to have high affinity for ABAT in the ileum) or cholylamidofluorescein (CAMF) (which has low affinity for ABAT)] and Texas Red-dextran as a fluid phase recovery marker. The percentage of ductal bile acid absorption was determined from the ratio of collected to perfusate fluorescence. Bile acid fluorescence was expressed as a ratio to Texas Red fluorescence to correct for dilution from IBDU secretion and potential leakage of fluorescent bile acid from the impaled sites. Cholangiocyte cAMP levels were increased by the addition of forskolin (10.4 M). RESULTS: IBDU luminal diameter (l00-200 /-Lm) remained unchanged during the perfusion experiments. The percentage of perfused C-L-NBD and CAMF absorbed by IBDU was 13% and 2% respectively. 77% of C-L-NBD absorption was dependent on the presence of Na+ in the perfusate. Forskolin increased Na+-dependent C-L-NBD absorption to 22% (p<0.05 compared to control) but had no effect on CAMF absorption. The absence of bicarbonate or CI' did not alter basal or forskolin-stimulated C-L-NBD absorption. C-L-NBD fluorescence did not accumulate in the cytoplasm of cholangiocytes suggesting apical bile acid uptake is the rate limiting step. SUMMARY/CONCLUSIONS: We have established a microperfused system for the study of bile acid absorption in IBDU. Consistent with an ABAT transport mechanism, ductal absorption is primarily Na+-dependent, showed substrate specificity identical to ABAT in the ileum, is not dependent on an anion exchange mechanism, and increases with elevated cholangiocyte cAMP levels. These data are consistent with our hypothesis that ABAT is the primary bile acid transporter in cholangiocytes.
152
Noriatsu Kanno, Shannon Glaser, Usha Chowdhury, Jo Lynne Phinizy, Heather Francis, Gene LeSage, Gianfranco Alpini, Texas A&M Univ, Temple, TX; Scott & White Hosp, Temple, TX; Texas A&M Univ and Cent Texas Veterans HSC, Temple, TX.
BIDIRECTIONAL WATER TRANSPORT ACROSS BILIARY EPI· THELIA.
Cholangiocytes are the target cells during hyperplastic and neoplastic growth in the liver. Cholangiocarcinoma, derived from intrahepatic and extrahepatic bile duct epithelial cells, displays a poor prognosis without established measures for treatment. In addition, the regulation of its growth is not fully defined. We have shown that the parasympathetic system plays an important role in the regulation of hyperplastic cholangiocyte growth and apoptosis. Adrenergic receptors regulate hyperplastic and neoplastic growth in a number of cell types through cAMP or 1P3-dependent mechanisms. No information exists regarding the role of the adrenergic system in the regulation of cholangiocarcinoma growth. AIMS: To evaluate the role and mechanisms of action of alpha-2 adrenergic receptors in the modulation of cholangiocarcinoma growth. METHODS: Studies were performed in the human cholangiocarcinoma cell line, Mz-ChA-l (a gift from Dr. G. Fitz). We first studied: (i) the expression of three subtypes (alpha2A, alpha-2B and alpha-2C) of adrenergic receptors in Mz-ChA-I cells by both immunocytochemistry and Western blotting analysis; and (ii) the effect of the alpha-2 adrenergic agonist (UK 14,304, 10 ILM) on the intracellular levels of cAMP and IP]. We determined the effect of UK 14,304 on cholangiocarcinoma DNA synthesis (evaluated by measurement of 3H-thymidine incorporation and PCNA protein expression) and apoptosis (determined by annexin-V staining) in the presence or absence of BAPTNAM (an intracellular calcium chelator), H7 (a Ca2i- -dependent PKCainhibitor), or rottlerin (a Ca2+-independent PKC-Ilinhibitor). RESULTS: Mz-ChA-l cells expressed alpha-2A, alpha-2B and alpha-2C receptors. UK14,304 inhibited the growth of Mz-ChA-1 cells in a doseand time-dependent manner. The inhibitory effect of UK 14,304 on cholangiocarcinoma growth was not affected by BAPT N AM, H7 or rottlerin but was associated with decreased levels of cAMP but not by IP 3. This inhibitory effect of UK14,304 was associated with increased apoptosis of Mz-ChA-l cells. SUMMARY/CONCLUSIONS: Mz-ChA-l cells expressed functional alpha-2 adrenergic receptors. The antiproliferative effect of UK 14,304 on cholangiocarcinoma growth was associated with increased apoptosis through the cAMP system. The data suggest that modulation of the adrenergic system could be exploited in the management of cholangiocarcinoma growth.
Progress in understanding ductal bile formation requires techniques that allow measurement of transepithelial water transport in intact bile ducts. We previously reported an experimental approach that allows microperfusion of the lumen of intrahepatic bile duct units (IBDUs) isolated from normal rat liver (Gastroenterology l16:A1236, 1999). Our AIM here was to study transepithelial water transport in perfused IBDUs using an epifiuorescence technique to provide quantitative water transport data in real time. METHODS. IBDUs (100-150 /-Lm diameter, 1-1.5 mm length) isolated from normal rat liver were perfused in vitro with a membraneimpermeant f1uorophore, fluorescein-sulfonate (FS), as a volume marker. Microperfused IBDUs were epiilluminated to excite FS fluorescence in a small spot (50-100 /-Lm) at the distal end of the lumen of IBDUs. Net water movement (Jv) and osmotic water permeability (Pf) were calculated from the FS concentration, perfusate rate flow, lumen and bath osmolalities, length of the IBDU, and surface area of the lumen. RESULTS. In the absence of an osmotic gradient (i.e., equal perfusate and bath buffer osmolality at 290 mOsm), the FS fluorescence was proportional to FS concentration, independent of perfusion flow rate, and constant along the length of perfused IBDUs. In contrast, when an inward osmotic gradient was established across IBDUs (perfusate osmolality-290 mOsm; bath osmolality-70 mOsm), the FS fluorescence at the distal end of the IBDU promptly (~30 sec) decr:ase~ indicati~§ water ~ov:ment ~om bath .to lumen, i.e., secretion (Pf-4.8_0.8 x 10 ern/sec; Jv--39.l_6.1 nl/mm/ mm). When a net outward osmotic gradient was established across IBDUs (perfusate osmolality-290 mOsm; bath osmolality-530 mOsm), the FS fluorescence promptly (~30 sec) increased, indicating water movement from lumen to bath, i.e., absorption (Pf=1.0:'::0.1 x 10_.3 ern/sec, Jv=24.l :'::2.7 nl/min/mm). Bath reperfusion of IBDUs with isotonic KRB returned FS fluorescence to its original level. CONCLUSION. Based on experimental models of water transport across other epithelial barriers, we developed a new experimental approach to study rapid changes in transepithelial water transport in intact bile ducts. The results indicate a relatively high Jv and Pf for IBDUs, providing functional evidence that intrahepatic bile ducts can both secrete and absorb water in response to osmotic gradients, presumably through molecular water channels.
Anatoly I. Masyuk, Michael J. Burke, Ai-Yu Gong, Nicholas F. LaRusso, Mayo Med Sch, Clin and Fdn, Rochester, MN.