Human hepatic cell uptake of resveratrol: involvement of both passive diffusion and carrier-mediated process

We evaluated the relationship between apical surface fluid (ASF) and protein secretion in Calu-3 cells grown at an air-liquid interface. Calu-3 monolayers responded to forskolin, a cystic fibrosis transmembrane regulator (CFTR) channel agonist, by secreting a significant amount of ASF. Such a response from Calu-3 monolayers was not observed with CFTR channel blockers glybenclamide and DPC. Other ion channel mediators, PGF-2alpha, PMA, DNDS, and DIDS, had no effect on Calu-3 ASF secretion. Forskolin decreased Calu-3 protein secretion and glybenclamide increased protein secretion. Similarly, forskolin decreased Calu-3 lysozyme secretion, whereas glybenclamide and DPC increased lysozyme secretion. We observed significant changes in Calu-3 fluid and protein secretions with ion channel mediators known to alter CFTR activity. Our results demonstrate a functional link between fluid and protein secretions in Calu-3 apical surface and suggested a possible involvement of CFTR in these processes.     

Involvement of cystic fibrosis transmembrane conductance regulator in infection-induced edema

Abnormal fluid accumulation in tissues, including the life-threatening cerebral and pulmonary edema, is a severe consequence of bacteria infection. Chlamydia (C.) trachomatis is an obligate intracellular gram-negative human pathogen responsible for a spectrum of diseases, causing tissue fluid accumulation and edema in various organs. However, the underlying mechanism for tissue fluid secretion induced by C. trachomatis and most of other infectious pathogens is not known. Here, we report that in mice C. trachomatis infection models, the expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel, is up regulated together with increased cytokine release and tissue fluid accumulation that can be reversed by treatment with antibiotic specific for C. trachomatis and CFTR channel blocker. However, C. trachomatis infection cannot induce tissue edema in CFTRtm1Unc mutant mice. Administration of exogenous IL-1beta to mice mimics the C. trachomatis infection-induced CFTR upregulation, enhanced CFTR channel activity and fluid accumulation, further confirming the involvement of CFTR in infection-induced tissue fluid secretion.     

Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel critical to intestinal anion secretion. In addition to phosphorylation, vesicle traffic regulates CFTR in some epithelial cells. Studies of cultured intestinal cells are conflicting regarding the role of cAMP-dependent vesicle traffic in regulating chloride transport. Whether CFTR is present in vesicular compartments within chloride secretory cells in the intestine is unknown and the role of cAMP-dependent vesicle insertion in regulating CFTR and intestinal fluid secretion remains unclear. The purpose of this study was to: (1) examine and quantify the subcellular distribution for CFTR in rat intestine, (2) further define the ultrastructure of the previously identified CFTR High Expresser (CHE) cell, and (3) examine the cellular distribution of CFTR following cAMP stimulation in vivo. Using the sensitive techniques of cryoimmunogold electron microscopy we identified CFTR in subapical vesicles and on the apical plasma membrane in crypt, Brunner glands, and CHE cells. cAMP stimulation in rat proximal small intestine produced a fluid secretory response and was associated with an apical redistribution of CFTR, supporting a physiologic role for cAMP-dependent CFTR vesicle insertion in regulating CFTR in the intestine.     

CFTR型氯离子通道研究进展

囊性纤维化跨膜传导调节因子（CFTR）是一种重要的氯离子通道,突变易引起囊性纤维化病变,故得名。一系列研究表明,CFTR由5个结构域组成：两个跨膜结构域形成氯离子通道;两个核苷酸结合结构域调节通道的开闭;一个调节结构域主要影响氯通道的活动。这些结构域通过协同作用共同控制了氯离子的跨膜流动,而一些突变可以影响细胞功能而导致囊性纤维化的发生。本文通过介绍CFTR基本结构、调节机制、与囊性纤维化病变的关系及针对CFTR的治疗而对CFTR型氯离子通道有一个的全面的理解。     

Hyperacidification of cellubrevin endocytic compartments and defective endosomal recycling in cystic fibrosis respiratory epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.     

Heterogeneity in the severity of cystic fibrosis and the role of CFTR gene mutations

Cystic fibrosis is a common, fatal disorder caused by abnormalities in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a chloride channel that regulates secretion in many exocrine tissues. The presentation of cystic fibrosis is highly variable as measured by the age of onset of disease, the presence of pancreatic insufficiency, or the progression of lung disease. Over 400 mutations in the CFTR gene have been described in cystic fibrosis patients and considerable effort has focused on the correlation between specific mutations and genotypes and clinical characteristics. Individual tissues display variation in their sensitivity to CFTR mutations. The vas deferens is functionally disrupted in nearly all males, whereas mild and severe pancreatic involvement is determined by the patient's genotype. The severity of pulmonary disease is poorly correlated with genotype, suggesting that there are other important genetic and/or environmental factors that contribute to lung infections and the subsequent disruption of lung function.     

cAMP-dependent exocytosis and vesicle traffic regulate CFTR and fluid transport in rat jejunum in vivo

The cystic fibrosis transmembraneconductance regulator (CFTR) channel is regulated by cAMP-dependentvesicle traffic and exocytosis to the apical membrane in some celltypes, but this has not been demonstrated in the intestinal crypt. Thedistribution of CFTR, lactase (control), and fluid secretion weredetermined in rat jejunum after cAMP activation in the presence ofnocodazole and primaquine to disrupt vesicle traffic. CFTR and lactasewere localized by immunofluorescence, and surface proteins weredetected by biotinylation of enterocytes. Immunoprecipitates frombiotinylated and nonbiotinylated cells were analyzed by streptavidindetection and immunoblots. Immunolocalization confirmed acAMP-dependent shift of CFTR but not lactase from a subapicalcompartment to the apical surface associated with fluid secretion thatwas reduced in the presence of primaquine and nocodazole. Analysis ofimmunoblots from immunoprecipitates after biotinylation revealed a3.8 ± 1.7-fold (P < 0.005) increase ofsurface-exposed CFTR after vasoactive intestinal peptide (VIP). Thesemeasurements provide independent corroboration supporting a role forvesicle traffic in regulating CFTR and cAMP-induced fluid transport inthe intestine.

     

Effects of CFTR gene silencing by siRNA or the luminal application of a CFTR activator on fluid secretion from guinea-pig pancreatic duct cells

Aims

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP regulated chloride channel expressed in the apical plasma membrane of pancreatic duct cells where it plays an important role in fluid secretion. The purpose of this study was to elucidate the role of the CFTR chloride channel on ion and fluid secretion from the guinea-pig pancreas by manipulating the expression of CFTR by RNA interference or by luminal application of a CFTR selective activator, MPB91, in isolated cultured pancreatic ducts.

Materials and methods

Using cDNA isolated from the guinea-pig small intestine, fragments of the CFTR gene were generated by polymerase chain reaction and directly sequenced. Two different RNA duplexes for small interference RNA (siRNA) were designed from the sequence obtained. Fluid secretion from the isolated guinea-pig pancreatic ducts was measured using video-microscopy. The amount of CFTR chloride channel or AQP1 water channel expressed in pancreatic ducts was examined by immunoblotting with antibodies against CFTR or AQP1, respectively.

Results

Guinea-pig CFTR consists of 1481 amino acid residues. An additional glutamine residue was found to be inserted between amino acid residues 403 and 404 of human CFTR. Forskolin-stimulated fluid secretion from intact pancreatic ducts was significantly higher in the presence of MPB91 compared to fluid secretion in the absence of MPB91. Both basal and forskolin-stimulated fluid secretion in pancreatic ducts transfected with CFTR specific siRNAs were reduced by ∼50% compared to fluid secretion from ducts transfected with scrambled negative control dsRNAs. The amount of CFTR and AQP1 proteins was reduced to 34% and 45% of control, respectively.

Conclusions

The activity of the CFTR chloride channel or the amount of CFTR protein expressed determines the rate of fluid secretion from the isolated guinea-pig pancreatic ducts.     

Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP

The CFTR [CF (cystic fibrosis) transmembrane conductance regulator] chloride channel is activated by cyclic nucleotide-dependent phosphorylation and ATP binding, but also by non-phosphorylation-dependent mechanisms. Other CFTR functions such as regulation of exocytotic protein secretion are also activated by cyclic nucleotide elevating agents. A soluble protein comprising the first NBD (nucleotide-binding domain) and R-domain of CFTR (NBD1-R) was synthesized to determine directly whether CFTR binds cAMP. An equilibrium radioligand-binding assay was developed, firstly to show that, as for full-length CFTR, the NBD1-R protein bound ATP. Half-maximal displacement of [3H]ATP by non-radioactive ATP at 3.5 microM and 3.1 mM was demonstrated. [3H]cAMP bound to the protein with different affinities from ATP (half-maximal displacement by cAMP at 2.6 and 167 microM). Introduction of a mutation (T421A) in a motif predicted to be important for cyclic nucleotide binding decreased the higher affinity binding of cAMP to 9.2 microM. The anti-CFTR antibody (MPNB) that inhibits CFTR-mediated protein secretion also inhibited cAMP binding. Thus binding of cAMP to CFTR is consistent with a role in activation of protein secretion, a process defective in CF gland cells. Furthermore, the binding site may be important in the mechanism by which drugs activate mutant CFTR and correct defective DeltaF508-CFTR trafficking.     

Synergistic effects of cystic fibrosis transmembrane conductance regulator and aquaporin-9 in the rat epididymis

The cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporin-9 (AQP-9) are present in the luminal membrane of the epididymis, where they play an important role in formation of the epididymal fluid. Evidence is accumulating that CFTR regulates other membrane transport proteins besides functioning as a cAMP-activated chloride channel. We have explored the possible interaction between epididymal CFTR and AQP-9 by cloning them from the rat epididymis and expressing them in Xenopus oocytes. The effects of the expressed proteins on oocyte water permeability were studied by immersing oocytes in a hypo-osmotic solution, and the ensuing water flow was measured using a gravimetric method. The results show that AQP-9 alone caused an increase in oocyte water permeability, which could be further potentiated by CFTR. This potentiation was markedly reduced by phloretin and lonidamine (inhibitors of AQP-9 and CFTR, respectively). The regulation of water permeability by CFTR was also demonstrated in intact rat epididymis luminally perfused with a hypo-osmotic solution. Osmotic water reabsorption across the epididymal tubule was reduced by phloretin and lonidamine. Elevation of intracellular cAMP with 3-isobutyl-1-methylxanthine increased osmotic water permeability, whereas inhibiting protein kinase A with H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline sulfonamide hydrochloride) reduced it. These results are consistent with a role for CFTR in controlling water permeability in the epididymis in vivo. We conclude that this additional role of CFTR in controlling water permeability may have an impact on the genetic disease cystic fibrosis, in which men with a mutated CFTR gene have abnormal epididymis and infertility.     

TMD2前断裂CFTR翻译后的连接及氯离子通道功能

CFTR基因突变导致一种常染色体隐性遗传疾病——囊性纤维化(CF)。利用split Ssp DnaB intein的蛋白质反式剪接技术的真核细胞双载体转CFTR基因,旨在研究翻译后水平CFTR的连接,以及由其建立的氯离子通道功能。于CFTR膜内第2个跨膜结构域(TMD2)前的Glu838密码子后将其cDNA断裂为N端和C端两部分,与具有蛋白质反式剪接作用的split Ssp DnaB intein编码序列融合,分别插入到载体pEGFP-N1和pEYFP-N1,构建一对真核表达载体pEGFP-NInt和pEYFP-IntC。用脂质体将这对载体共转染至幼年仓鼠肾细胞(BHK),瞬时表达实验用Western blotting观察CFTR蛋白质的连接,并用膜片钳技术记录Cl-通道电流。结果显示,基因共转染细胞呈现完整的CFTR蛋白条带,膜片钳记录到全细胞Cl-电流和单个Cl-通道开放活性。结果表明split Ssp DnaB intein的蛋白质反式剪接技术可用于双载体共转移CFTR基因,为CF基因治疗应用双腺相关病毒载体(AAV)转运CFTR基因,克服AAV的容量限制提供了依据。     

STa and cGMP stimulate CFTR translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase G

The cystic fibrosis transmembrane conductance regulator (CFTR) is critical to cAMP- and cGMP-activated intestinal anion secretion and the pathogenesis of secretory diarrhea. Enterotoxins released by Vibrio cholerae (cholera toxin) and Escherichia coli (heat stable enterotoxin, or STa) activate intracellular cAMP and cGMP and signal CFTR on the apical plasma membrane of small intestinal enterocytes to elicit chloride and fluid secretion. cAMP activates PKA, whereas cGMP signals a cGMP-dependent protein kinase (cGKII) to phosphorylate CFTR in the intestine. In the jejunum, cAMP also regulates CFTR and fluid secretion by insertion of CFTR from subapical vesicles to the surface of enterocytes. It is unknown whether cGMP signaling or phosphorylation regulates the insertion of CFTR associated vesicles from the cytoplasm to the surface of enterocytes. We used STa, cell-permeant cGMP, and cAMP agonists in conjunction with PKG and PKA inhibitors, respectively, in rat jejunum to examine whether 1) cGMP and cGK II regulate the translocation of CFTR to the apical membrane and its relevance to fluid secretion, and 2) PKA regulates cAMP-dependent translocation of CFTR because this intestinal segment is a primary target for toxigenic diarrhea. STa and cGMP induced a greater than fourfold increase in surface CFTR in enterocytes in association with fluid secretion that was inhibited by PKG inhibitors. cAMP agonists induced a translocation of CFTR to the cell surface of enterocytes that was prevented by PKA inhibitors. We conclude that cAMP and cGMP-dependent phosphorylation regulates fluid secretion and CFTR trafficking to the surface of enterocytes in rat jejunum. small intestine; cystic fibrosis transmembrane conductance regulator; membrane traffic; phosphorylation     

Ca2+-activated Cl- channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion.

This study addresses the mechanisms by which a defect in CFTR impairs pancreatic duct bicarbonate secretion in cystic fibrosis. We used control (PANC-1) and CFTR-deficient (CFPAC-1; DeltaF508 mutation) cell lines and measured HCO3- extrusion by the rate of recovery of intracellular pH after an alkaline load and recorded whole cell membrane currents using patch clamp techniques. 1) In PANC-1 cells, cAMP causes parallel activation of Cl- channels and of HCO3- extrusion by DIDS-sensitive and Na+-independent Cl-/HCO3- exchange, both effects being inhibited by Cl- channel blockers NPPB and glibenclamide. 2) In CFPAC-1 cells, cAMP fails to stimulate Cl-/HCO3- exchange and Cl- channels, except after promoting surface expression of DeltaF508-CFTR by glycerol treatment. Instead, raising intracellular Ca2+ concentration to 1 micromol/l or stimulating purinergic receptors with ATP (10 and 100 micromol/l) leads to parallel activation of Cl- channels and HCO3- extrusion. 3) K+ channel function is required for coupling cAMP- and Ca2+-dependent Cl- channel activation to effective stimulation of Cl-/HCO3- exchange in control and CF cells, respectively. It is concluded that stimulation of pancreatic duct bicarbonate secretion via Cl-/HCO3- exchange is directly correlated to activation of apical membrane Cl- channels. Reduced bicarbonate secretion in cystic fibrosis results from defective cAMP-activated Cl- channels. This defect is partially compensated for by an increased sensitivity of CF cells to purinergic stimulation and by alternative activation of Ca2+-dependent Cl- channels, mechanisms of interest with respect to possible treatment of cystic fibrosis and of related chronic pancreatic diseases.     

Non-specific activation of the epithelial sodium channel by the CFTR chloride channel

The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of ²²Na⁺ through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride channels. Voltage– and patch–clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of ²²Na⁺ uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR.     

Murine colonic mucosa hyperproliferation. I. Elevated CFTR expression and enhanced cAMP-dependent Cl(-) secretion

Fluid transport in the large intestine is mediated by the cystic fibrosis gene product and cAMP-dependent anion channel cystic fibrosis transmembrane conductance regulator (CFTR). cAMP-mediated Cl(-) secretion by gastrointestinal cell lines in vitro has been positively correlated with the insertion of CFTR into the apical membrane of differentiated senescent colonocytes and negatively correlated with the failure of CFTR to insert into the plasma membrane of their undifferentiated proliferating counterparts. In native tissues, this relationship remains unresolved. We demonstrate, in a transmissible murine colonic hyperplasia (TMCH) model, that (8-fold) colonocyte proliferation was accompanied by increased cellular CFTR mRNA and protein expression (8.3- and 2.4-fold, respectively) and enhanced mucosal cAMP-dependent Cl(-) secretion (2. 3-fold). By immunofluorescence microscopy, cellular CFTR expression was restricted to the apical pole of cells at the base of the epithelial crypt. In contrast, increased cellular proliferation in vivo led to increases in both the cellular level and the total number of cells expressing this anion channel, with cellular CFTR staining extending into the crypt neck region. Hyperproliferating colonocytes accumulated large amounts of CFTR in apically oriented subcellular perinuclear compartments. This novel mode of CFTR regulation may explain why high endogenous levels of cellular CFTR mRNA and protein within the TMCH epithelium were not matched with larger increases in transmucosal CFTR Cl(-) current.     

A mutation in the cystic fibrosis transmembrane conductance regulator generates a novel internalization sequence and enhances endocytic rates

Cystic fibrosis is a common lethal genetic disease among Caucasians. The cystic fibrosis gene encodes a cyclic adenosine monophosphate-activated chloride channel (cystic fibrosis transmembrane conductance regulator (CFTR)) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. Mutations in CFTR fall into two broad categories; those that affect protein biosynthesis/stability and traffic to the cell surface and those that cause altered channel kinetics in proteins that reach the cell surface. Here we report a novel mechanism by which mutations in CFTR give rise to disease. N287Y, a mutation within an intracellular loop of CFTR, increases channel endocytosis from the cell surface without affecting either biosynthesis or channel gating. The sole consequence of this novel mutation is to generate a novel tyrosine-based endocytic sequence within an intracellular loop in CFTR leading to increased removal from the cell surface and a reduction in the steady-state level of CFTR at the cell surface.     

Activation of CFTR by ASBT-mediated bile salt absorption

In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal ileum might also modulate intestinal fluid secretion. Taurocholate (TC) induced a biphasic rise in the short circuit current across ileal tissue, reflecting transepithelial electrogenic ion transport. This response was sensitive to bumetanide and largely abrogated in Cftr-null mice, indicating that it predominantly reflects CFTR-mediated Cl- secretion. The residual response in Cftr-null mice could be attributed to electrogenic ASBT activity, as it matched the TC-coupled absorptive Na+ flux. TC-evoked Cl- secretion required ASBT-mediated TC uptake, because it was blocked by a selective ASBT inhibitor and was restricted to the distal ileum. Suppression of neurotransmitter or prostaglandin release, blocking of the histamine H1 receptor, or pretreatment with 5-hydroxytryptamine did not abrogate the TC response, suggesting that neurocrine or immune mediators of Cl- secretion are not involved. Responses to TC were retained after carbachol treatment and after permeabilization of the basolateral membrane with nystatin, indicating that BS modulate CFTR channel gating rather than the driving force for Cl- exit. TC-induced Cl- secretion was maintained in cGMP-dependent protein kinase II-deficient mice and only partially inhibited by the cAMP-dependent protein kinase inhibitor H89, suggesting a mechanism of CFTR activation different from cAMP or cGMP signaling. We conclude that active BS absorption in the ileum triggers CFTR activation and, consequently, local salt and water secretion, which may serve to prevent intestinal obstruction in the postprandial state.     

Model of the cAMP activation of chloride transport by CFTR channel and the mechanism of potentiators

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis, a hereditary lethal disease. CFTR is a chloride channel expressed in the apical membrane of epithelia. It is activated by cAMP dependent phosphorylation and gated by the binding of ATP. The impaired chloride transport of some types of cystic fibrosis mutations could be pharmacologically solved by the use of chemical compounds called potentiators. Here it is undertaken the construction of a model of the CFTR activation pathways, and the possible modification produced by a potentiator application. The model yields a novel mechanism for the potentiator action, describing the activatory and inhibitory activities on two different positions in the CFTR activation pathway.     

The cystic fibrosis and is product CFTR

Cystic fibrosis is the commonest, fatal, inherited disease of caucasian populations occurring with a frequency of 1 in 2000 live births. The CF gene spans about 230 kb of genomic DNA and encodes a protein of 1480 amino acids named the cystic fibrosis transmembrane conductance regulator (CFTR). The primary sequence predicts that CFTR is an ABC type protein with twelve transmembrane spans, two nucleotide binding domains and a cytoplasmic regulatory domain. CFTR functions as a cyclic AMP-regulated, low conductance, chloride channel in epithelial cells, but other roles are possible. Failure of the CFTR channel in CF reduces epithelial salt and water secretion, leading to a dehydration of epithelial surfaces which initiates the pathology of the disease.