Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines

Abstract

We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into human intrahepatic biliary epithelial (IBE) cells from three different normal individuals. These IBE cell lines grow in a hormone-supplemented medium in the presence of NIH/3T3 fibroblast coculture. These cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratins 7 and 19 and gamma-glutamyl transpeptidase while being negative for the hepatocyte markers albumin and asialoglycoprotein receptor. To evaluate ion transport pathways in IBE cell lines, we utilized intracellular pH (pHi) measurements obtained using the intracellular fluorescent indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In the absence of HCO3(-)-CO2, an amiloride-sensitive Na(+)-H+ exchanger participated in the regulation of basal pHi. In the presence of HCO3(-)-CO2, a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive, Na-, Cl-, and HCO3(-)-dependent acid extrusion mechanism accounted for approximately 60% of pHi recovery from acidic pHi; this mechanism is most consistent with the presence of a Na-dependent Cl-HCO3- exchanger (Na+HCO3(-)-Cl-H+). Under basal conditions, Cl- depletion revealed a DIDS-sensitive alkalinization consistent with a Na-independent Cl(-)-HCO3- exchanger. These model systems will allow the opportunity to study the normal mechanisms of IBE function and to study the pathobiology of IBE processes in disease states.

Expression of mediators of purinergic signaling in human liver cell lines

“…Human H69 and Mz-ChA-1 cholangiocyte-derived cells H69 cell line [38] was developed through retroviral SV40 transformation of human cholangiocytes, while Mz-ChA-1 cell line [36] is derived from mechanically dissociated gallbladder adenocarcinoma metastases. All adenosine receptors were shown to be expressed by both H69 and MzChA-1 cell lines (Fig.…”

“…Cell lines and culture conditions LX-2 [33] [38] (kindly provided by Dr. Doug Jefferson, Tufts University School of Medicine, Boston, MA) were cultured, as previously described [38], in a mixture (1:1, volume/volume) of DMEM and DMEM/F12 media containing 10 % FBS, 1 % antibiotics, 1 % adenine, 0.125 % insulin, 0.1 % epinephrine, 0.1 % triiodothyronine-transferrin, 0.33 % epidermal growth factor, and 0.267 % hydrocortisone. All cell cultures were maintained at 37°C with a 5.0 % CO 2 , humidified environment, and passaged every 2-3 days.…”

A biliary HCO3−umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes

“…The human cholangiocarcinoma cell lines, TFK-1 and EGI-1, were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Lonza, Basel, Switzerland) with 4 mM of glutamine and 10% fetal calf serum (FCS) or Dulbecco's minimal essential medium (MEM) (Lonza) with 4 mM of glutamine, 10% FCS, and MEM essential and nonessential amino acids (Gibco, Carlsbad, CA), respectively. The immortalized nonmalignant human intrahepatic cholangiocyte cell line, H69, 35 was cultured in a DMEM/F12 (3:1)-based medium, enriched with 4 mM of glutamine, 180 lM of adenin, 865 nM of insulin, 10 lg/L of epidermal growth factor, 5 mg/L of triiodothyronin, 1.1 lM of hydrocortisone, and 5 mg/L of transferrin. All media contained 40 U/mL of penicillin, 40 ug/mL of streptomycin, and 0.1 ug/mL of amphotericin, and cells were kept at 37 C in a 5% CO 2 atmosphere and passaged twice-weekly.…”

The Human Immunodeficiency Virus Type 1 Tat Protein EnhancesCryptosporidium parvum-Induced Apoptosis in Cholangiocytes via a Fas Ligand-Dependent Mechanism

“…The human bile duct epithelial cell line H69 is a simian virus 40-transformed cell line that was originally derived from a normal liver harvested for transplantation. H69 cells have been extensively characterized and continue to express cholangiocyte markers consistent with biliary function (20). Cells between passages 20 and 30 were used for this study.…”