We previously found that water transport across hepatocyte plasma membranes occurs mainly via a nonchannel mediated pathway. Recently, it has been reported that mRNA for the water channel, aquaporin-8 (AQP8), is present in hepatocytes. To further explore this issue, we studied protein expression, subcellular localization, and regulation of AQP8 in rat hepatocytes. By subcellular fractionation and immunoblot analysis, we detected an N-glycosylated band of ϳ34 kDa corresponding to AQP8 in hepatocyte plasma and intracellular microsomal membranes. Confocal immunofluorescence microscopy for AQP8 in cultured hepatocytes showed a predominant intracellular vesicular localization. Dibutyryl cAMP (Bt 2 cAMP) stimulated the redistribution of AQP8 to plasma membranes. Bt 2 cAMP also significantly increased hepatocyte membrane water permeability, an effect that was prevented by the water channel blocker dimethyl sulfoxide. The microtubule blocker colchicine but not its inactive analog lumicolchicine inhibited the Bt 2 cAMP effect on both AQP8 redistribution to cell surface and hepatocyte membrane water permeability. Our data suggest that in rat hepatocytes AQP8 is localized largely in intracellular vesicles and can be redistributed to plasma membranes via a microtubule-depending, cAMP-stimulated mechanism. These studies also suggest that aquaporins contribute to water transport in cAMP-stimulated hepatocytes, a process that could be relevant to regulated hepatocyte bile secretion.Bile is formed primarily by hepatocytes and subsequently delivered to the bile ducts where it is modified by cholangiocytes (i.e. the epithelial cells that line the bile ducts). Bile secretion by hepatocytes involves the active transport of solutes followed by the passive movement of water into the bile canaliculus in response to osmotic gradients created by these solutes (1, 2). Although a substantial amount of data have been published about the molecular identification of solute transporters and the mechanisms regulating solute transport by hepatocytes (3), little attention has been focused on the mechanistic and regulatory aspects involved in hepatocyte water transport.Water can cross cellular plasma membranes through the lipid portion of the bilayer by a diffusion mechanism or through aquaporin water channels. Aquaporins, a family of recently identified integral membrane proteins, increase cell membrane water permeability facilitating rapid movement of water in response to osmotic gradients (4, 5).We previously found based on biophysical and molecular biology studies that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway (6). As this observation seems to be in contradiction with the recent identification of transcript for the water channel aquaporin-8 (AQP8) 1 in hepatocytes (7-9), we further explored this issue by studying the protein expression, subcellular localization, and possible regulation of AQP8 water channels in isolated rat hepatocytes. MATERIALS AND METHODSIsolation and Incubation of ...

Although bile formation requires that large volumes of water be rapidly transported across liver epithelia, including hepatocytes, the molecular mechanisms by which water is secreted into bile are obscure. The aquaporins are a family of 10 channel-forming, integral membrane proteins of ϳ28 kDa numbered 0 -9 that allow water to rapidly traverse epithelial barriers in several organs including kidney, eye, and brain. We found transcripts of three of 10 aquaporins in hepatocytes (aquaporin 8 > > aquaporin 9 > aquaporin 0) by reverse transcription-polymerase chain reaction and quantitative ribonuclease protection assays; immunohistochemistry confirmed the presence of these three proteins in liver. Immunoblots of subcellular fractions of hepatocytes showed enrichment of aquaporins 0 and 8 in microsomes and canalicular plasma membranes; aquaporin 9 was enriched only in basolateral plasma membranes. Immunofluorescence of hepatocyte couplets confirmed the intracellular/canalicular localization of aquaporins 0 and 8 and the basolateral localization of aquaporin 9. Upon exposure of couplets to a choleretic stimulus (i.e. dibutyryl cAMP), aquaporin 8 redistributed to the canalicular plasma membrane; the subcellular distributions of aquaporins 0 and 9 were unaffected. In addition, exposure of couplets to dibutyryl cAMP caused an increase in canalicular water transport in the presence and absence of an osmotic gradient, an effect that was blocked by aquaporin inhibitors. These results provide evidence that aquaporins are present in hepatocytes and that aquaporins are involved in agonist-stimulated canalicular bile secretion.Primary bile is secreted by hepatocytes at the bile canaliculus and is modified via absorption and secretion of water, ions, and solutes by cholangiocytes, the epithelial cells that line the intrahepatic bile ducts. Hepatocytes are polarized epithelial cells that possess well defined canalicular and basolateral plasma membranes and are capable of rapidly transporting large volumes of water (1). Bile consists of 99% water, and water transport by hepatocytes is thought to occur passively in response to local, transient, osmotic gradients generated by the active transport of osmotically active solutes, especially bile acids (2). Two pathways exist by which water could potentially move from blood to bile across the hepatocyte epithelial barrier: a paracellular pathway through the tight junctions between adjacent hepatocytes and a transcellular pathway across hepatocytes. Furthermore, transcellular water movement across individual hepatocytes could theoretically occur either by diffusion through the lipid portion of the sinusoidal and canalicular hepatocyte plasma membranes or through aquaporin water channels, proteins that span the plasma membrane and allow for bi-directional, passive flux of water in response to soluteinduced osmotic gradients. The quantitative contributions of these potential pathways (i.e. paracellular versus transcellular) and mechanisms (i.e. diffusion versus channel-mediated) of water tr...

Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear.

Although secretin is known to stimulate ductal bile secretion by directly interacting with cholangiocytes, the precise cellular mechanisms accounting for this choleretic effect are unknown. We have previously shown that secretin stimulates exocytosis in cholangiocytes and that these cells transport water mainly via the water channel aquaporin-1 (AQP1). In this study, we tested the hypothesis that secretin promotes osmotic water movement in cholangiocytes by inducing the exocytic insertion of AQP1 into plasma membranes. Exposure of highly purified isolated rat cholangiocytes to secretin caused significant, dose-dependent increases in osmotic membrane water permeability (P f ) (e.g. increased by 60% with 10 ؊7 M secretin), which was reversibly inhibited by the water channel blocker HgCl 2 . Immunoblotting analysis of cholangiocyte membrane fractions showed that secretin caused up to a 3-fold increase in the amount of AQP1 in plasma membranes and a proportional decrease in the amount of the water channel in microsomes, suggesting a secretin-induced redistribution of AQP1 from intracellular to plasma membranes. Both the secretin-induced increase in cholangiocyte P f and AQP1 redistribution were blocked by two perturbations that inhibit secretin-stimulated exocytosis in cholangiocytes, i.e. treatment with colchicine and exposure at low temperatures (20 and 4°C). Our results demonstrate that secretin increases AQP1-mediated P f in cholangiocytes. Moreover, our studies implicate the microtubule-dependent vesicular translocation of AQP1 water channels to the plasma membrane, a mechanism that appears to be essential for secretin-induced ductal bile secretion and suggests that AQP1 can be regulated by membrane trafficking.Bile is formed primarily by hepatocytes and secreted at the bile canaliculus; subsequently, its volume and composition are modified in the lumen of bile ducts as a result of the transport of water and solutes by cholangiocytes (1, 2). While this ductal bile secretion results from the osmotically driven movement of water, the regulatory and mechanistic aspects are obscure. We recently reported that cholangiocytes (unlike hepatocytes) express the water-selective channel protein aquaporin-1 (AQP1) 1 and proposed that ductal bile secretion results from the movement of water across this protein (3, 4). Based on studies in renal epithelial cells, it is currently thought that AQP1 is constitutively inserted into plasma membranes and is not hormone responsive (5, 6).Secretin is known to stimulate ductal bile secretion via specific receptors on cholangiocytes (7). We and others recently proposed that secretin-induced bile secretion was associated with the microtubule-dependent exocytic insertion of cytoplasmic vesicles into the cholangiocyte plasma membrane (8 -10). Interestingly, hormone-regulated exocytic movement of transporters has been demonstrated in other cell types (11). For example, in renal collecting tubule cells the water channel aquaporin-2 moves to and from the apical plasma membrane in the presence...

Aquaporin-1 (AQP1) water channels are present in the apical and basolateral plasma membrane domains of bile duct epithelial cells, or cholangiocytes, and mediate the transport of water in these cells. We previously reported that secretin, a hormone known to stimulate ductal bile secretion, increases cholangiocyte osmotic water permeability and stimulates the redistribution of AQP1 from an intracellular vesicular pool to the cholangiocyte plasma membrane. Nevertheless, the target plasma membrane domain (i.e., basolateral or apical) for secretin-regulated trafficking of AQP1 in cholangiocytes is unknown, as is the functional significance of this process for the secretion of ductal bile. In this study, we used primarily an in vivo model (i.e., rats with cholangiocyte hyperplasia induced by bile duct ligation) to address these issues. AQP1 was quantitated by immunoblotting in apical and basolateral plasma membranes prepared from cholangiocytes isolated from rats 20 min after intravenous infusion of secretin. Secretin increased bile flow (78%, P < 0.01) as well as the amount of AQP1 in the apical cholangiocyte plasma membrane (127%, P < 0.05). In contrast, the amount of AQP1 in the basolateral cholangiocyte membrane and the specific activity of an apical cholangiocyte marker enzyme (i.e., γ-glutamyltranspeptidase) were unaffected by secretin. Similar observations were made when freshly isolated cholangiocytes were directly exposed to secretin. Immunohistochemistry for AQP1 in liver sections from secretin-treated rats showed intensified staining at the apical region of cholangiocytes. Pretreatment of rats with colchicine (but not with its inactive analog β-lumicolchicine) inhibited both the increases of AQP1 in the cholangiocyte plasma membrane (94%, P < 0.05) and the bile flow induced by secretin (54%, P < 0.05). Our results in vivo indicate that secretin induces the microtubule-dependent insertion of AQP1 exclusively into the secretory pole (i.e., apical membrane domain) of rat cholangiocytes, a process that likely accounts for the ability of secretin to stimulate ductal bile secretion.

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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...

Overall, these results experimentally prove major functional significance for AQP9 in maximising liver glycerol import during states requiring increased glucose production. If any, alternative facilitated pathways would be of minor importance in transporting glucogenetic glycerol into hepatocytes during starvation. Refining the understanding of liver AQP9 in metabolic and energy homeostasis may reveal helpful for therapeutic purposes.