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Background & Aims: The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. Methods: The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. Results: Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced d-myo-inositol 1,4,5-triphosphate/Ca^2^+/protein kinase C signaling and the consequent inhibition of the adenosine 3',5'-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. Conclusions: We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies.

Background & Aims: We evaluated the expression of neurotrophins in rat cholangiocytes and the role and mechanisms by which nerve growth factor (NGF) modulates cholangiocyte proliferation. Methods: The expression of neurotrophins and their receptors was investigated by immunohistochemistry in liver sections and reverse-transcription polymerase chain reaction and immunoblots in isolated cholangiocytes. In vitro, the effect of NGF on cholangiocyte proliferation and signal transduction was investigated by immunoblotting for proliferating cell nuclear antigen, phosphorylated AKT (p-AKT), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated c-jun-N-terminal kinase, and phosphorylated p38. In vivo, rats that had undergone bile duct ligation (BDL) were treated with an anti-NGF antibody to immunoneutralize NGF and bile duct mass, proliferation, apoptosis, and inflammation were investigated by immunohistochemistry. Results: NGF and its TrkA receptor were expressed by normal rat cholangiocytes and up-regulated following BDL. Cholangiocytes secrete NGF, and secretion is increased in proliferating BDL cholangiocytes. In vitro, NGF stimulated cholangiocyte proliferation, which was associated with enhanced p-AKT and p-ERK1/2 expression. NGF proliferation in vitro was partially blocked by the MEK inhibitor (UO126) and completely ablated by the phosphatidylinositol 3-kinase inhibitor (wortmannin). In vitro, NGF and estrogens have an additive effect on cholangiocyte proliferation by acting on phosphorylated TrkA and p-ERK1/2. In vivo, immunoneutralization of NGF decreased bile duct mass in BDL rats, which was associated with depressed proliferation and enhanced apoptosis and with increased portal inflammation. Conclusions: Cholangiocytes secrete NGF and express NGF receptors. NGF induces cholangiocyte proliferation by activating the ERK and, predominantly, the phosphatidylinositol 3-kinase pathway and exerts an additive effect in combination with estrogens on proliferation.

Background & Aims:: We investigated the expression of glucocorticoid receptors (GcRs) in the intrahepatic biliary epithelium and the role of corticosteroids in the regulation of cholangiocyte secretion. Methods:: GcR was studied by immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blots. The effects of dexamethasone and budesonide on biliary bicarbonate excretion and H^+/HCO"3^- transport processes were investigated in bile fistula rats, isolated intrahepatic bile duct units (IBDUs), and purified cholangiocytes. Results:: GcRs were expressed by rat cholangiocytes. Although acute administration of corticosteroids showed no effect, treatment for 2 days with dexamethasone or budesonide increased (P < 0.05) biliary bicarbonate concentration and secretion, which were blocked by the specific GcR antagonist, RU-486. IBDUs isolated from rats treated with dexamethasone or budesonide showed an increased (P < 0.05) activity of the Na^+/H^+ exchanger (NHE1 isoform) and Cl^-/HCO"3^- exchanger (AE2 member), which was blocked by RU-486. Protein expression of NHE1 and AE2 and messenger RNA for NH1 but not AE2 were increased (P < 0.05) in isolated cholangiocytes by dexamethasone treatment. Conclusions:: The intrahepatic biliary epithelium expresses GcR and responds to corticosteroids by increasing bicarbonate excretion in bile. This is caused by corticosteroid-induced enhanced activities and protein expression of transport processes driving bicarbonate excretion in the biliary epithelium.

Background & Aims: To investigate the role of the cholinergic system in regulation of cholangiocyte functions, we evaluated the effects of vagotomy on cholangiocyte proliferation and secretion in rats that underwent bile duct ligation (BDL rats). Methods: After bile duct ligation (BDL), the vagus nerve was resected; 7 days later, expression of M3 acetylcholine receptor was evaluated. Cholangiocyte proliferation was assessed by morphometry and measurement of DNA synthesis. Apoptosis was evaluated by light microscopy and annexin-V staining. Ductal secretion was evaluated by measurement of secretin-induced choleresis, secretin receptor (SR) gene expression, and cyclic adenosine 3',5'-monophosphate (cAMP) levels. Results: Vagotomy decreased the expression of M3 acetylcholine receptors in cholangiocytes. DNA synthesis and ductal mass were markedly decreased, whereas cholangiocyte apoptosis was increased by vagotomy. Vagotomy decreased ductal secretion. Forskolin treatment prevented the decrease in cAMP levels induced by vagotomy, maintained cholangiocyte proliferation, and decreased cholangiocyte apoptosis caused by vagotomy in BDL rats. Cholangiocyte secretion was also maintained by forskolin. Conclusions: Vagotomy impairs cholangiocyte proliferation and enhances apoptosis, leading to decreased ductal mass in response to BDL. Secretin-induced choleresis of BDL rats was virtually eliminated by vagotomy in association with decreased cholangiocyte cAMP levels. Maintenance of cAMP levels by forskolin administration prevents the effects of vagotomy on cholangiocyte proliferation, apoptosis, and secretion. GASTROENTEROLOGY 1999;117:191-199