Na+-dependent and -independent Cl/HCOmath image exchange mediate cellular HCOmath image transport in cultured human intrahepatic bile duct cells

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Abstract

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCOmath image and Cl. Mechanisms responsible for transepithelial H+/HCOmath imagesecretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, γ-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2′7′-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCOmath image, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na+-dependent and amiloride-inhibitable. No Na+-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCOmath image, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl-depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na+-dependent and Na+-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCOmath imageexchange activity was not prevented by 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-/HCOmath imageexchange) and an acid loader (Cl-/HCOmath imageexchange), whereas no evidence was found for cAMP activated H+-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl/HCOmath imageexchange, whereas Na+:HCOmath image cotransport is not active in the physiological range of pHi. Stimulation of Na+-independent Cl/HCOmath imageexchanger by cAMP does not require activation of Clconductances. These mechanisms may underlay hormone-regulated biliary HCOmath imagesecretion in the human biliary tree.

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