Agonist-induced Coordinated Trafficking of Functionally Related Transport Proteins for Water and Ions in Cholangiocytes

Abstract

We previously proposed that ductal bile formation is regulated by secretin-responsive relocation of aquaporin 1 (AQP1), a water-selective channel protein, from an intracellular vesicular compartment to the apical membrane of cholangiocytes. In this study, we immunoisolated AQP1-containing vesicles from cholangiocytes prepared from rat liver; quantitative immunoblotting revealed enrichment in these vesicles of not only AQP1 but also cystic fibrosis transmembrane regulator (CFTR) and AE2, a Cl ؊ channel and a Cl ؊ /HCO 3 ؊ exchanger, respectively. Dual labeled immunogold electron microscopy of cultured polarized mouse cholangiocytes showed significant colocalization of AQP1, CFTR, and AE2 in an intracellular vesicular compartment; exposure of cholangiocytes to dibutyryl-cAMP (100 M) resulted in co-redistribution of all three proteins to the apical cholangiocyte plasma membrane. After administration of secretin to rats in vivo, bile flow increased, and AQP1, CFTR, and AE2 co-redistributed to the apical cholangiocyte membrane; both events were blocked by pharmacologic disassembly of microtubules. Based on these in vitro and in vivo observations utilizing independent and complementary approaches, we propose that cholangiocytes contain an organelle that sequesters functionally related proteins that can account for ion-driven water transport, that this organelle moves to the apical cholangiocyte membrane in response to secretory agonists, and that these events account for ductal bile secretion at a molecular level.Cholangiocytes are cells that line intrahepatic bile ducts and, like other epithelia, possess discrete, specialized apical and basolateral membranes. Each cholangiocyte membrane contains specific receptors and flux molecules (i.e. channels, exchangers, and transporters) that accomplish the vectorial movement of solutes, ions, and water across the biliary epithelial barrier (1-3), resulting in ductal bile formation by as yet unclear molecular mechanisms. Recently, we proposed a molecular model for hormone-induced bile secretion by cholangiocytes. The key feature of this model is the agonist-induced, coordinated, exocytic insertion into and endocytic retrieval from the apical cholangiocyte plasma membrane of key flux molecules that, in the unstimulated state, are sequestered in an intracytoplasmic vesicular compartment (4). We also proposed that one of these flux proteins was AQP1, a member of the aquaporin (AQP) 1 family of water channels that mediate the bidirectional, passive movement of water molecules across epithelial cells in response to osmotic gradients established by ions and solutes (1, 5-7). In support of this model are data showing that secretin, a hormone that stimulates ductal bile secretion, can also trigger the exocytic insertion of AQP1 into the apical cholangiocyte plasma membrane (6). More recently, we also provided data in hepatocytes, the other epithelial cell in the liver involved in bile formation, indicating that recycling of AQP8 may account for agonist-induced canalicular bile secreti...

Shared apical sorting of anion exchanger isoforms AE2a, AE2b1, and AE2b2 in primary hepatocytes

“…The distribution of GFP signals in the subapical vesicular compartment, and former reports on a detrimental effect of colchicine on bicarbonate-stimulated AE activity in rat hepatocytes [3], suggested that targeting of AE2 isoforms may involve microtubule-associated vesicular transport and exocytotic insertion in the canalicular membrane. This view is also supported by recent evidence in cholangiocytes for the presence of AE2 in subapical vesicles (together with two functionally related proteins, i.e., the chloride channel CFTR and the water channel AQP1), that move in a microtubule-dependent manner to the apical membrane in response to secretory agonists [30]. Thus, in accordance with a participation of microtubules in AE2 distribution, we found that treatment with colchicine partially dispersed the fluorescent signals from GFP-tagged AE2 isoforms, and resulted in an extensive punctate fluorescence across the cytoplasm, the green fluorescence being often detected at the basolateral membrane as well (Fig.…”

Secretin activation of the apical Na+-dependent bile acid transporter is associated with cholehepatic shunting in rats

“…These data are consistent with previous studies showing that secretin increases vesicle traffic toward the cholangiocyte apical membrane and increases exocytosis in cholangiocytes. 10,11 Recent studies have shown that secretin increases cholangiocyte aquaporin and CFTR by microtubule-dependent insertion of aquaporins and CFTR into the cholangiocyte apical membrane. 10,11 Since CFTR and aquaporins translocate together in the same vesicle population, 10,11 the translocation of ASBT may be associated with other cholangiocyte apical membrane transporters.…”

“…10,11 Recent studies have shown that secretin increases cholangiocyte aquaporin and CFTR by microtubule-dependent insertion of aquaporins and CFTR into the cholangiocyte apical membrane. 10,11 Since CFTR and aquaporins translocate together in the same vesicle population, 10,11 the translocation of ASBT may be associated with other cholangiocyte apical membrane transporters. Hormonestimulated insertion of transporters in renal tubule cells and intestinal epithelial cells regulate secretion.…”

“…9 Aquaporin channels and cystic fibrosis transmembrane conductor regulation (CFTR) insertion onto the apical membrane of cholangiocytes are associated with increases of water and Cl Ϫ transport in cholangiocytes. 10,11 In these studies, we experimentally tested whether ASBT translocates to the cholangiocyte apical membrane and if increased ASBT activity (due to translocation) results in enhanced cholehepatic shunting of conjugated bile acids. Immunofluorescence microscopy and subcellular fractionation studies show that ASBT is intracellular.…”

Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger

“…More recently, AE2 was described to colocalize in cholangiocytes with CFTR and the water channel aquaporin-1 in intracellular vesicles, which co-redistribute to the apical membrane upon both cAMP and secretin stimulation. 19 Although AE2 appears to be a good candidate as the effector of the Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchange in cholangiocytes, the possible role of the remaining transporter polypeptides has never been ruled out, and the putative physiological role for AE2 is awaiting definite demonstration.…”