Division of Gene Therapy and Hepatology-Liver Unit, CIMA, CUN, and School of Medicine, University of Navarra, and Ciberehd, Pamplona, Spain.
Hepatology. 2010 Mar;51(3):891-902. doi: 10.1002/hep.23403.
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.
Bicarbonate secretion in mouse cholangiocytes involves two differentially regulated activities: Ae2-mediated Cl(-)/HCO(3) (-) exchange and Na(+)-HCO(3) (-) cotransport.
胆管细胞的碳酸氢盐分泌对于沿胆管适当调整初级胆小管胆汁是必需的。在人和大鼠胆管细胞中，碳酸氢盐分泌由阴离子交换器 (AE) 2 介导，AE 2 是一种电中性的 Na(+)-独立的 Cl(-)/HCO(3)(-)AE，也参与细胞内 pH (pH(i))调节。在 Ae2(a,b)-缺陷小鼠中，淋巴细胞和成纤维细胞中的 pH(i)增加，而胆管细胞中的 pH(i)出人意料地正常。在这里，我们通过对已建立的灌注操作过程中 pH(i)变化的微荧光测量，分析培养的 Ae2(a,b)(+/+)和 Ae2(a,b)(-/-)小鼠胆管细胞中 HCO(3)(-)分泌的机制。基于异丁酸盐的灌注中 Cl(-)的去除表明，Ae2(a,b)(+/+)但不是 Ae2(a,b)(-/-)小鼠胆管细胞可以显示 Cl(-)/HCO(3)(-)交换，因此完全由 Ae2 介导。然而，同时去除 Cl(-)和 Na(+)表明，小鼠胆管细胞具有额外的 HCO(3)(-)分泌转运活性，而在对照大鼠胆管细胞中未观察到这种活性。基于丙酸盐的操作表明，这种补充的 Na(+)-驱动的 HCO(3)(-)分泌活性是 Cl(-)独立的，与 Na(+)-HCO(3)(-)共转运体 (NBC)一致。与 Ae2(a,b)(-/-)小鼠胆管细胞中的 NBC 活性大于 Ae2(a,b)(+/+)相比，NBC 活性更大，并且膜去极化实验表明它是电致的。与 Slc4a4/Nbc1 作为涉及的转运蛋白的潜在作用一致，Ae2(a,b)(-/-)小鼠胆管细胞表现出这种电致 NBC 载体的上调表达。虽然 Ae2 介导的 Cl(-)/HCO(3)(-)交换在 Ae2(a,b)(+/+)小鼠胆管细胞中被环磷酸腺苷 (cAMP)和乙酰胆碱刺激，但 NBC 活性在 Ae2(a,b)(-/-)小鼠胆管细胞中被 cAMP 和三磷酸腺苷 (ATP)下调。放置在 Ussing 室中的极化 Ae2(a,b)(-/-)小鼠胆管细胞显示出减少的 (但未消除的) cAMP 依赖性 Cl(-)电流和增加的 ATP 依赖性/钙 (Ca(2+))激活 Cl(-)分泌，这与囊性纤维化跨膜电导调节剂信使 RNA 表达的减少和细胞内 Ca(2+)水平的增加平行。
小鼠胆管细胞中的碳酸氢盐分泌涉及两种不同调节的活性：Ae2 介导的 Cl(-)/HCO(3)(-)交换和 Na(+)-HCO(3)(-)共转运。
