Gene LeSage

Ursodeoxycholic acid in the treatment of primary biliary cirrhosis

BACKGROUND/AIMS A double-blind, placebo-controlled trial of ursodeoxycholic acid (UDCA) was conducted in 180 patients with primary biliary cirrhosis (PBC) to define the efficacy and safety of UDCA. Efficacy was assessed by time to treatment failure defined as death; liver transplantation; histological progression; development of varices, ascites, or encephalopathy; doubling of total serum bilirubin levels; progression of fatigue or pruritus; drug toxicity; or voluntary withdrawal. METHODS Patients with well-defined PBC underwent complete history, physical examination, liver chemistries, ultrasonography, upper endoscopy, and liver biopsy at entry as well as at 2 years. Liver chemistries were determined every 3 months. RESULTS In patients receiving UDCA, treatment failure was delayed compared with the placebo-treated group (P = 0.0003, log rank test). Seven patients receiving UDCA died or required transplantation compared with 12 in the placebo group (P = 0.18). No patients discontinued UDCA because of side effects of toxicity. CONCLUSIONS UDCA was extraordinarily safe and well tolerated, and its use was associated with delayed progression of the disease as defined in this study. However, the lack of effects on symptoms, histology, and the need for liver transplantation or survival indicate that further evaluation is necessary to determine the ultimate role of UDCA in the treatment of PBC.

Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation

We previously introduced the concept that intrahepatic bile duct epithelial cells, or cholangiocytes, are functionally heterogeneous. This concept is based on the observation that secretin receptor (SR) gene expression and secretin-induced cAMP synthesis are present in cholangiocytes derived from large (> 15 microns in diameter) but not small (< 15 microns in diameter) bile ducts. In work reported here, we tested the hypothesis that cholangiocytes are heterogeneous with regard to proliferative capacity. We assessed cholangiocyte proliferation in vivo by measurement of [3H]thymidine incorporation and in vitro by both [3H]thymidine incorporation and H3 histone gene expression in small (fraction 1) and large (fraction 2) cholangiocytes isolated from rats after bile duct ligation (BDL). In the two cholangiocyte subpopulations, we also studied basal somatostatin receptor (SSTR2) gene expression as well as the effects of somatostatin on 1) SR gene expression and secretin-induced cAMP synthesis and 2) [3H]thymidine incorporation and H3 histone gene expression. In normal rat liver, cholangiocytes, unlike hepatocytes, were mitotically dormant; after BDL, incorporation of [3H]thymidine markedly increased in cholangiocytes but not hepatocytes. When subpopulations of cholangiocytes were isolated after BDL, DNA synthesis assessed by both techniques was limited to large cholangiocytes, as was SSTR2 steady-state gene expression. In vitro, somatostatin inhibited SR gene expression and secretin-induced cAMP synthesis only in large cholangiocytes. Moreover, compared with no hormone, somatostatin inhibited DNA synthesis solely in large cholangiocytes. These results support the concept of the heterogeneity of cholangiocytes along the biliary tree, extend this concept to cholangiocyte proliferative activity, and imply that the proliferative compartment of cholangiocytes after BDL is located principally in the cholangiocytes lining large (> 15 microns) bile ducts.We previously introduced the concept that intrahepatic bile duct epithelial cells, or cholangiocytes, are functionally heterogeneous. This concept is based on the observation that secretin receptor (SR) gene expression and secretin-induced cAMP synthesis are present in cholangiocytes derived from large (>15 μm in diameter) but not small (<15 μm in diameter) bile ducts. In work reported here, we tested the hypothesis that cholangiocytes are heterogeneous with regard to proliferative capacity. We assessed cholangiocyte proliferation in vivo by measurement of [3H]thymidine incorporation and in vitro by both [3H]thymidine incorporation and H3 histone gene expression in small ( fraction 1) and large ( fraction 2) cholangiocytes isolated from rats after bile duct ligation (BDL). In the two cholangiocyte subpopulations, we also studied basal somatostatin receptor (SSTR2) gene expression as well as the effects of somatostatin on 1) SR gene expression and secretin-induced cAMP synthesis and 2) [3H]thymidine incorporation and H3 histone gene expression. In normal rat liver, cholangiocytes, unlike hepatocytes, were mitotically dormant; after BDL, incorporation of [3H]thymidine markedly increased in cholangiocytes but not hepatocytes. When subpopulations of cholangiocytes were isolated after BDL, DNA synthesis assessed by both techniques was limited to large cholangiocytes, as was SSTR2 steady-state gene expression. In vitro, somatostatin inhibited SR gene expression and secretin-induced cAMP synthesis only in large cholangiocytes. Moreover, compared with no hormone, somatostatin inhibited DNA synthesis solely in large cholangiocytes. These results support the concept of the heterogeneity of cholangiocytes along the biliary tree, extend this concept to cholangiocyte proliferative activity, and imply that the proliferative compartment of cholangiocytes after BDL is located principally in the cholangiocytes lining large (>15 μm) bile ducts.

Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver

Bile duct damage and/or loss is limited to a range of duct sizes in cholangiopathies. We tested the hypothesis that CCl4damages only large ducts. CCl4 or mineral oil was given to bile duct-ligated (BDL) rats, and 1, 2, and 7 days later small and large cholangiocytes were purified and evaluated for apoptosis, proliferation, and secretion. In situ, we measured apoptosis by morphometric and TUNEL analysis and the number of small and large ducts by morphometry. Two days after CCl4 administration, we found an increased number of small ducts and reduced number of large ducts. In vitro apoptosis was observed only in large cholangiocytes, and this was accompanied by loss of proliferation and secretion in large cholangiocytes and loss of choleretic effect of secretin. Small cholangiocytes de novo express the secretin receptor gene and secretin-induced cAMP response. Consistent with damage of large ducts, we detected cytochrome P-4502E1 (which CCl4 converts to its radicals) only in large cholangiocytes. CCl4induces selective apoptosis of large ducts associated with loss of large cholangiocyte proliferation and secretion.

Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice

Aims: We have shown that large and small cholangiocytes, which reside primarily in large and small intrahepatic bile ducts, respectively, have different functions and responses to injuries. However, there are no systematic studies of the molecular differences between small and large cholangiocytes, which would explain cholangiocyte heterogeneity. To evaluate the differential gene expression between small and large cholangiocytes, microarray analysis was performed.

Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2+-dependent protein kinase C-α

BACKGROUND/AIMS We determined the role of gastrin in the regulation of cholangiocarcinoma growth. METHODS We evaluated for the functional presence of cholecystokinin (CCK)-B/gastrin receptors in the cholangiocarcinoma cell lines, Mz-ChA-1, HuH-28 and TFK-1. We determined the effect of gastrin on the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. We evaluated the effect of gastrin on growth and apoptosis of Mz-ChA-1 in the absence or presence of inhibitors for CCK-A (L-364, 718) and CCK-B/gastrin (L-365, 260) receptors, the intracellular Ca2+ chelator (BAPTA/AM), and the protein kinase C (PKC)-alpha inhibitor, H7. We evaluated if gastrin effects on Mz-ChA-1 growth and apoptosis are associated with membrane translocation of PKC-alpha. RESULTS Gastrin inhibited DNA synthesis of Mz-ChA-1, HuH-28 and TFK-1 cells in a dose- and time-dependent fashion. The antiproliferative effect of gastrin on Mz-ChA-1 cells was inhibited by L-365, 260, H7 and BAPTA/AM but not L-364, 718. Gastrin induced membrane translocation of PKC-alpha. The inhibition of growth of Mz-ChA-1 cells by gastrin was associated with increased apoptosis through a PKC-dependent mechanism. CONCLUSIONS Gastrin inhibits the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. Gastrin inhibits growth and induces apoptosis in Mz-ChA-1 cells through the Ca2+-dependent PKC-alpha. The data suggest a therapeutic role for gastrin in the modulation of cholangiocarcinoma growth.

α-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca2+- and PKC-dependent stimulation of cAMP

Acetylcholine potentiates secretin‐stimulated ductal secretion by Ca2+‐calcineurin–mediated modulation of adenylyl cyclase. D2 dopaminergic receptor agonists inhibit secretin‐stimulated ductal secretion via activation of protein kinase C (PKC)‐γ. No information exists regarding the effect of adrenergic receptor agonists on ductal secretion in a model of cholestasis induced by bile duct ligation (BDL). We evaluated the expression of α‐1A/1C, ‐1β and β‐1 adrenergic receptors in liver sections and cholangiocytes from normal and BDL rats. We evaluated the effects of the α‐1 and β‐1 adrenergic receptor agonists (phenylephrine and dobutamine, respectively) on bile and bicarbonate secretion and cholangiocyte IP3 and Ca2+ levels in normal and BDL rats. We measured the effect of phenylephrine on lumen expansion in intrahepatic bile duct units (IBDUs) and cyclic adenosine monophosphate (cAMP) levels in cholangiocytes from BDL rats in the absence or presence of BAPTA/AM and Gö6976 (a PKC‐α inhibitor). We evaluated if the effects of phenylephrine on ductal secretion were associated with translocation of PKC isoforms leading to increased protein kinase A activity. α‐1 and β‐1 adrenergic receptors were present mostly in the basolateral domain of cholangiocytes and, following BDL, their expression increased. Phenylephrine, but not dobutamine, increased secretin‐stimulated choleresis in BDL rats. Phenylephrine did not alter basal but increased secretin‐stimulated IBDU lumen expansion and cAMP levels, which were blocked by BAPTA/AM and Gö6976. Phenylephrine increased IP3 and Ca2+ levels and activated PKC‐α and PKC‐β‐II. In conclusion, coordinated regulation of ductal secretion by secretin (through cAMP) and adrenergic receptor agonist activation (through Ca2+/PKC) induces maximal ductal bicarbonate secretion in liver diseases. (Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:1116–1127))

Regulation of cholangiocyte bile secretion

he intrahepatic biliary system is comprised of aset of anastomosing and progressively largertubes resembling a tree lined by cholangiocytes (1,2).The major function of the biliary system is modifi-cation of canalicular bile by secretory and reabsorptiveprocesses in cholangiocytes as bile passes through bileducts before reaching the duodenum (1,3–8). In con-trast to hepatocytes, where secretion is primarily un-regulated and constant (9), bile duct secretion is modi-fied by hormones, peptides, nerves and biliary con-stituents (1,3–8,10–24). The bile duct system attractsattention from clinicians, pathologists, physiologistsand cell biologists because it is the target for a varietyof liver diseases including primary biliary cirrhosis(PBC), primary sclerosing cholangitis (PSC), graft

Secretin activation of the apical Na+-dependent bile acid transporter is associated with cholehepatic shunting in rats

The role of the cholangiocyte apical Na+‐dependent bile acid transporter (ASBT) in bile formation is unknown. Bile acid absorption by bile ducts results in cholehepatic shunting, a pathway that amplifies the canalicular osmotic effects of bile acids. We tested in isolated cholangiocytes if secretin enhances ASBT translocation to the apical membrane from latent preexisting intracellular stores. In vivo, in bile duct–ligated rats, we tested if increased ASBT activity (induced by secretin pretreatment) results in cholehepatic shunting of bile acids. We determined the increment in taurocholate‐dependent bile flow and biliary lipid secretion and taurocholate (TC) biliary transit time during high ASBT activity. Secretin stimulated colchicine‐sensitive ASBT translocation to the cholangiocyte plasma membrane and 3H‐TC uptake in purified cholangiocytes. Consistent with increased ASBT promoting cholehepatic shunting, with secretin pretreatment, we found TC induced greater‐than‐expected biliary lipid secretion and bile flow and there was a prolongation of the TC biliary transit time. Colchicine ablated secretin pretreatment‐dependent bile acid–induced choleresis, increased biliary lipid secretion, and the prolongation of the TC biliary transit. In conclusion, secretin stimulates cholehepatic shunting of conjugated bile acids and is associated with increased cholangiocyte apical membrane ASBT. Bile acid transport by cholangiocyte ASBT can contribute to hepatobiliary secretion in vivo. (HEPATOLOGY 2005.)

Essential role of toll-like receptor 2 in morphine-induced microglia activation in mice

Opioids are powerful pain relievers, but also potent inducers of dependence and tolerance. Chronic morphine administration (via subcutaneous pellet) induces morphine dependence in the nucleus accumbens, an important dependence region in the brain, yet the cellular mechanisms are mostly unknown. Toll-like receptor 2 (TLR2) plays an essential function in controlling innate and inflammatory responses. Using a knockout mouse lacking TLR2, we assessed the contribution of TLR2 to microglia activation and development of morphine dependence. We report here that mice deficient in TLR2 inhibit morphine-induced the levels of microglia activation and proinflammatory cytokines. Moreover, in TLR2 knockout mice the main symptoms of morphine withdrawal were significantly attenuated. Our data reveal that TLR2 plays a critical role in morphine-induced microglia activation and dependence.

AF17 Competes with AF9 for Binding to Dot1a to Up-regulate Transcription of Epithelial Na+ Channel α

We previously reported that Dot1a·AF9 complex represses transcription of the epithelial Na+ channel subunit α (α-ENaC) gene in mouse inner medullary collecting duct mIMCD3 cells and mouse kidney. Aldosterone relieves this repression by down-regulating the complex through various mechanisms. Whether these mechanisms are sufficient and conserved in human cells or can be applied to other aldosterone-regulated genes remains largely unknown. Here we demonstrate that human embryonic kidney 293T cells express the three ENaC subunits and all of the ENaC transcriptional regulators examined. These cells respond to aldosterone and display benzamil-sensitive Na+ currents, as measured by whole-cell patch clamping. We also show that AF17 and AF9 competitively bind to the same domain of Dot1a in multiple assays and have antagonistic effects on expression of an α-ENaC promoter-luciferase construct. Overexpression of Dot1a or AF9 decreased mRNA expression of the ENaC subunits and their transcriptional regulators and reduced benzamil-sensitive Na+ currents. AF17 overexpression caused the opposite effects, accompanied by redirection of Dot1a from the nucleus to the cytoplasm and reduction in histone H3 K79 methylation. The nuclear export inhibitor leptomycin B blocked the effect of AF17 overexpression on H3 K79 hypomethylation. RNAi-mediated knockdown of AF17 yielded nuclear enrichment of Dot1a and histone H3 K79 hypermethylation. As with AF9, AF17 displays nuclear and cytoplasmic co-localization with Sgk1. Therefore, AF17 competes with AF9 to bind Dot1a, decreases Dot1a nuclear expression by possibly facilitating its nuclear export, and relieves Dot1a·AF9-mediated repression of α-ENaC and other target genes.