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...
The CFTR splicing mutation 3849 ؉ 10 kb C 3 T creates a novel donor site 10 kilobases (kb) into intron 19 of the gene and is one of the more common splicing mutations that causes cystic fibrosis (CF). It has an elevated prevalence among patients with atypically mild disease and normal sweat electrolytes and is especially prominent in Ashkenazi Jews. This class of splicing mutations, reported in several genes, involves novel splice sites activated deep within introns while leaving wild-type splice elements intact. CFTR cDNA constructs that modeled the 3849 ؉ 10 kb C 3 T mutation were expressed in 3T3 mouse fibroblasts and in CFT1 human tracheal and C127 mouse mammary epithelial cells. In all three cell types, aberrant splicing of CFTR pre-mRNA was comparable to that reported in vivo in CF patients. Treatment of the cells with 2-O-methyl phosphorothioate oligoribonucleotides antisense toward the aberrant donor and acceptor splice sites or to the retained exon-like sequence, disfavored aberrant splicing and enhanced normal processing of CFTR pre-mRNA. This antisense-mediated correction of splicing was dose-and sequencedependent and was accompanied by increased production of CFTR protein that was appropriately glycosylated. Antisense-mediated correction of splicing in a mutation-specific context represents a potential gene therapy modality with applicability to many inherited disorders. Cystic fibrosis (CF)1 is an inherited disorder characterized by multi-organ involvement and substantial heterogeneity in the presentation of disease (1). At a molecular level, the pathogenesis of CF is attributable in part to over 800 known mutations 2 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which interfere with the processing or integrity of the CFTR protein, a cAMP-activated chloride channel (3-6). To the extent that mutations within the CFTR gene permit residual chloride channel activity, a milder phenotype typically results.Approximately 14% of the deleterious mutations known to cause cystic fibrosis interfere with mRNA splicing, a frequency comparable to that reported for other inherited disorders (3, 7).2 Of the splicing mutations reported, the great majority disrupt either the splice acceptor or splice donor sites that demarcate the 5Ј and 3Ј ends of each exon, respectively, and drive the exclusion of that exon from the mature transcript.Splicing may also be derailed by mutations within introns that create novel splice sites, resulting in the inappropriate inclusion of non-coding sequence. This often occurs close to exons, but may also occur deep within introns, creating either a novel donor or acceptor site that, in conjunction with a nearby cryptic splice site of the opposite polarity, defines a novel, aberrant exon that the spliceosome recognizes and includes into the mature message. Several examples of this mutational mechanism have been shown to underlie inherited diseases, such as -thalassemia (8 -12), CF (13,14), neurofibromatosis type 1 (15), multiple breast tumors (16), dihydropter...
Most messenger RNA precursors (pre-mRNA) undergo cissplicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its premRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conduc-
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