NA-DEPENDENT AND NA+-INDEPENDENT CL-()HCO3- EXCHANGE MEDIATE CELLULARHCO3- TRANSPORT IN CULTURED HUMAN INTRAHEPATIC BILE-DUCT CELLS/

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and a lkalinity absorbing and/or secreting fluid and electrolytes, particula rly HCO3- and Cl-. Mechanisms responsible for transepithelial H+/HCO3 secretion in human cholangiocytes are largely unknown. Human cholangio cytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatr ic liver transplantation were cultured in the presence of human hepato cyte growth factor. Maintenance of cholangiocyte phenotypic features w as assessed using markers such as cytokeratin 19, gamma-glutamyltransp eptidase, vimentin, factor VIII-related antigen, desmin, epithelial me mbrane antigen (EMA), and human epithelial antigen (HEA) 125, Intracel lular pH (pHi) transients were measured microfluorimetrically 2'7'-Bis (2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3- pHi recovery from an intracellular acid load ( ammonia pre-pulse technique) was Na+-dependent and amiloride-inhibitab le. No Na+-independent recovery was recorded even after stimulation wi th agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3- recovery from an intracellula r acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4,-diisothiocyan a tostilben-2,2,-disulfonic acid (DIDS) and by intracellular Cl- depleti on. Acute removal of extracellular Cl- induced a pHi alkalinization th at was inhibited by DIDS. pHi recovery hom an intracellular alkaline l oad (isohydric CO2 changes) was Cl--dependent and DIDS-inhibitable. Ad ministration of agents raising intracellular cAMP concentrations incre ased both Na+-dependent and Na+-independent Cl(-)MCO(3)(-) exchange ac tivity, Stimulation of Cl-/HCO3- exchange activity was not prevented b y the Cl- channel inhibitor 5'-nitro-2(2)-phenylpropyl-amino-benzoate (NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+ exchanger and Na+-dependent Cl-/HCO3- exchange) and an acid l oader (Cl-/HCO3- exchange), whereas no evidence was found for cAMP act ivated H+-ATPase. Bicarbonate influx is thus mainly mediated by Na-dep endent Cl-/HCO3- exchange, whereas Na+:HCO3 cotransport is not active in the physiological range of pHi. Stimulation of Na+-independent Cl-/ HCO3 exchanger by cAMP does not require activation of Cl- conductances . These mechanisms may underlay hormone-regulated biliary HCO3- secret ion in the human biliary tree.