We sought to develop a cholangiocyte cell culture system that has preservation of receptors, transporters, and channels involved in secretin-induced secretion. Isolated bile duct fragments, obtained ...by enzyme perfusion of normal rat liver, were seeded on collagen and maintained in culture up to 18 wk. Cholangiocyte purity was assessed by staining for -glutamyl transpeptidase (-GT) and cytokeratin-19 (CK-19). We determined gene expression for secretin receptor (SR), cystic fibrosis transmembrane conductance regulator, Cl/HCO exchanger, secretin-stimulated cAMP synthesis, Cl/HCO3 exchanger activity, secretin-stimulated Cl efflux, and apical membrane-directed secretion in polarized cells grown on tissue culture inserts. Cultured cholangiocytes were all -GT and CK-19 positive. The cells expressed SR and Cl/HCO exchanger, and secretin-stimulated cAMP synthesis, Cl/HCO exchanger activity, and Cl efflux were similar to freshly isolated cholangiocytes. Forskolin (104 M) induced fluid accumulation in the apical chamber of tissue culture inserts. In conclusion, we have developed a novel cholangiocyte line that has persistent HCO, Cl, and fluid transport functions. This cell system should be useful to investigators who study cholangiocyte secretion.
Growth factor signaling, mediated by the mitogen-activated protein kinase (MAPK) cascade, induces cell mitosis. Adenosine 3',5'-monophosphate (cAMP) may inhibit or stimulate mitosis (depending on the ...cell type) through the activation of MAPK and Raf proteins. Among Raf proteins, Raf-1 and B-Raf differentially regulate mitosis. Our aims were to evaluate the role and mechanisms of action of the α
2-adrenergic agonist UK14,304 in the regulation of growth of the human cholangiocarcinoma cell line Mz-ChA-1. Immunocytochemistry and immunoblotting for α
2A-, α
2B-, or α
2C-adrenergic receptor subtypes showed positive reaction in Mz-ChA-1 cells. We found that physiological concentrations of UK14,304 increased cAMP levels and inhibited proliferation and MAPK activity in Mz-ChA-1 cells. Mz-ChA-1 cells expressed Raf-1 and B-Raf. Epidermal growth factor (EGF) immediately and transiently stimulated Raf-1 activity, whereas B-Raf activity was increased with prolonged EGF stimulation. EGF-stimulated Raf-1 and B-Raf activities were both inhibited by UK14,304. UK14,304 did not affect Ras activity. In Mz-ChA-1 cells, α
2-adrenoreceptor stimulation causes up-regulation of cAMP, which inhibits EGF-induced MAPK activity through an acute increase of Raf-1 and sustained activation of B-Raf. In conclusion, because α
2-AR inhibition of growth occurred downstream of Ras, adrenergic stimulation or other stimulants of cAMP may overcome the Ras mutations and offer a new therapeutic approach for patients with cholangiocarcinoma. (H
EPATOLOGY 2002;35:1329-1340.)
Growth factor signaling, mediated by the mitogen-activated protein kinase (MAPK) cascade, induces cell mitosis. Adenosine 3',5'-monophosphate (cAMP) may inhibit or stimulate mitosis (depending on the ...cell type) through the activation of MAPK and Raf proteins. Among Raf proteins, Raf-1 and B-Raf differentially regulate mitosis. Our aims were to evaluate the role and mechanisms of action of the alpha(2)-adrenergic agonist UK14,304 in the regulation of growth of the human cholangiocarcinoma cell line Mz-ChA-1. Immunocytochemistry and immunoblotting for alpha(2A)-, alpha(2B)-, or alpha(2C)-adrenergic receptor subtypes showed positive reaction in Mz-ChA-1 cells. We found that physiological concentrations of UK14,304 increased cAMP levels and inhibited proliferation and MAPK activity in Mz-ChA-1 cells. Mz-ChA-1 cells expressed Raf-1 and B-Raf. Epidermal growth factor (EGF) immediately and transiently stimulated Raf-1 activity, whereas B-Raf activity was increased with prolonged EGF stimulation. EGF-stimulated Raf-1 and B-Raf activities were both inhibited by UK14,304. UK14,304 did not affect Ras activity. In Mz-ChA-1 cells, alpha(2)-adrenoreceptor stimulation causes up-regulation of cAMP, which inhibits EGF-induced MAPK activity through an acute increase of Raf-1 and sustained activation of B-Raf. In conclusion, because alpha(2)-AR inhibition of growth occurred downstream of Ras, adrenergic stimulation or other stimulants of cAMP may overcome the Ras mutations and offer a new therapeutic approach for patients with cholangiocarcinoma.
PDF
