Bicarbonate secretion of mouse cholangiocytes involves Na(+)-HCO(3)(-) cotransport in addition to Na(+)-independent Cl(-)/HCO(3)(-) exchange

Abstract

Bicarbonate secretion from cholangiocytes is required for appropriate adjustment of primary canalicular bile along the biliary tract. In human and rat cholangiocytes, bicarbonate secretion is mediated by anion exchanger (AE) 2, an electroneutral Na(+)-independent Cl(-)/HCO(3) (-) AE also involved in intracellular pH (pH(i)) regulation. In Ae2(a,b)-deficient mice, pH(i) is increased in lymphocytes and fibroblasts, whereas it is surprisingly normal in cholangiocytes. Here, we analyze the mechanisms for HCO(3) (-) secretion in cultured Ae2(a,b) (+/+) and Ae2(a,b) (-/-) mouse cholangiocytes by microfluorimetric measurement of pH(i) changes upon established perfusion maneuvers. Cl(-) withdrawal by isethionate-based perfusions showed that Ae2(a,b) (+/+) but not Ae2(a,b) (-/-) mouse cholangiocytes can display Cl(-)/HCO(3) (-) exchange, which is therefore entirely mediated by Ae2. Nevertheless, simultaneous withdrawal of Cl(-) and Na(+) revealed that mouse cholangiocytes possess an additional transport activity for HCO(3) (-) secretion not observed in control rat cholangiocytes. Propionate-based maneuvers indicated that this supplemental Na(+)-driven HCO(3) (-)-secreting activity is Cl(-)-independent, consistent with a Na(+)-HCO(3) (-) cotransport (NBC). NBC activity is greater in Ae2(a,b) (-/-) than Ae2(a,b) (+/+) mouse cholangiocytes, and membrane-depolarization experiments showed that it is electrogenic. Consistent with the potential role of Slc4a4/Nbc1 as the involved transporter, Ae2(a,b) (-/-) mouse cholangiocytes exhibit up-regulated expression of this electrogenic NBC carrier. Whereas Ae2-mediated Cl(-)/HCO(3) (-) exchange in Ae2(a,b) (+/+) mouse cholangiocytes is stimulated by cyclic adenosine monophosphate (cAMP) and acetylcholine, the NBC activity is down-regulated by cAMP and adenosine triphosphate (ATP) in Ae2(a,b) (-/-) mouse cholangiocytes. Polarized Ae2(a,b) (-/-) mouse cholangiocytes placed in Ussing chambers show decreased (but not abolished) cAMP-dependent Cl(-) current and increased ATP-dependent/Ca(2+)-activated Cl(-) secretion, which run in parallel with decreased cystic fibrosis transmembrane conductance regulator messenger RNA expression and increased intracellular Ca(2+) levels. Conclusion: Bicarbonate secretion in mouse cholangiocytes involves two differentially regulated activities: Ae2-mediated Cl(-)/HCO(3) (-) exchange and Na(+)-HCO(3) (-) cotransport.