Role Of Cystic Fibrosis Transmembrane Conductance Regulator Research Articles

ENaC- and CFTR-dependent ion and fluid transport in human middle ear epithelial cells

Ion channels, such as the epithelial sodium channel (ENaC), are essential for maintaining a fluid-free middle ear cavity by controlling periciliary fluid. Deviations from the normal volume or compositions of periciliary fluid are probably responsible for otitis media with effusion. To elucidate the physiologic roles of the ENaC and cystic fibrosis transmembrane conductance regulator (CFTR) in the middle ear mucosa, we compared the electrophysiological activity and protein expressions of ENaC and CFTR in normal human middle ear epithelial (NHMEE) cells with those in normal human nasal epithelial (NHNE) cells. We also evaluated the role of ENaC and CFTR in fluid transport by NHMEE cells. Short-circuit current ( I sc) was measured in cell monolayers by modified Ussing chambers. Immunoblotting was performed for ENaC and CFTR. In addition, transepithelial fluid transport was measured after loading 100 μl of fluid onto the luminal cell surface. The amiloride-sensitive Isc in NHMEE cells was much larger than in NHNE cells, whereas the forskolin-induced I sc, presumably mediated by CFTR, was significantly smaller in NHMEE cells. ENaC subunits α, β, and γ were all detected in NHMEE cells, and their expressions were stronger than those in NHNE cells. In comparison, CFTR was also detected in the middle ear mucosa, but at a lower expression level than in NHNE cells. NHMEE cells showed more amiloride-sensitive fluid absorption than NHNE cells. In contrast, fluid absorption was less sensitive to forskolin/IBMX in NHMEE cells than in NHNE cells. The ATP induced Cl − efflux and the amplitude of ATP-induced current in NHMEE cells was much larger than in NHNE cells. In the present study, we have demonstrated an enhanced amiloride-sensitive I sc and fluid absorption in NHMEE cells, where the role of CFTR is limited. Our data also suggest that the ATP-induced Cl − channel could be an alternative Cl − channel to CFTR in NHMEE cells.

Dynamic Control of Cystic Fibrosis Transmembrane Conductance Regulator [formula omitted] Selectivity by External Cl–

HCO(3)(-) secretion is a vital activity in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelia. However, the role of CFTR in this activity is not well understood. Simultaneous measurements of membrane potential and pH(i) and/or current in CFTRexpressing Xenopus oocytes revealed dynamic control of CFTR Cl(-)/HCO(3)(-) permeability ratio, which is regulated by external Cl(-) (Cl(-)(o)). Thus, reducing external Cl(-) from 110 to 0-10 mm resulted in the expected increase in membrane potential, but with no corresponding OH(-) or HCO(3)(-) influx. Approximately 3-4 min after reducing Cl(o)(-) to 0 mm, an abrupt switch in membrane potential occurs that coincided with an increased rates of OH(-) and HCO(3)(-) influx. The switch in membrane permeability to OH(-)/HCO(3)(-) can also be recorded as a leftward shift in the reversal potential. Furthermore, an increased rate of OH(-) influx in response to elevating pH(o) to 9.0 was observed only after the switch in membrane potential. The time to switch increased to 11 min at Cl(o)(-) of 5 mm. Conversely, re-addition of external Cl(-) after the switch in membrane potential did not stop HCO(3)(-) influx, which continued for about 3.9 min after Cl(-) addition. Importantly, addition of external Cl(-) to cells incubated in Cl(-)-free medium never resulted in HCO(3)(-) efflux. Voltage and current clamp experiments showed that the delayed HCO(3)(-) transport is electrogenic. These results indicate that CFTR exists in two conformations, a Cl(-) only and a Cl(-) and OH(-)/HCO(3)(-) permeable state. The switch between the states is controlled by external Cl(-). Accordingly, a different tryptic pattern of CFTR was found upon digestion in Cl(-)-containing and Cl(-)-free media. The physiological significance of these finding is discussed in the context of HCO(3)(-) secretion by tissues such as the pancreas and salivary glands.

Retinal pigment epithelial function: a role for CFTR?

In the vertebrate eye, the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE) are separated by a small extracellular (subretinal) space whose volume and chemical composition varies in the light and dark. Light onset triggers relatively fast (ms) retinal responses and much slower voltage and resistance changes (s to min) at the apical and basolateral membranes of the RPE. Two of these slow RPE responses, the fast oscillation (FO) and the light peak, are measured clinically as part of the electrooculogram (EOG). Both EOG responses are mediated in part by apical and basolateral membranes proteins that form a pathway for the movement of salt and osmotically obliged fluid across the RPE, from retina to choroid. This transport pathway serves to control the volume and chemical composition of the subretinal and choroidal extracellular spaces. In human fetal RPE, we have identified one of these proteins, the cystic fibrosis transmembrane conductance regulator (CFTR) by RT-PCR, immunolocalization, and electrophysiological techniques. Evidence is presented to suggest that the FO component of the EOG is mediated directly or indirectly by CFTR.

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.

Xenograft model of the CF airway.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in defective ion transport, leading to thick mucus, impaired mucociliary clearance and decreased bacterial killing in the lung (,). Despite recent progress in associating cystic fibrosis (CF) defects with CFTR dysfunction, there are many unanswered questions concerning the roles of CFTR in both normal airway biology and in CF pathology. Before effective therapeutic approaches for CF lung disease will be realized, several remaining challenges must first be overcome. These include a more precise definition of CFTR functions in fluid transport, electrolyte balance, and in the regulation of other epithelial ion channels. Moreover, the heterogeneity of CFTR expression among different cell types and regions of the lung necessitates the identification of pathophysiologic relevant cellular targets for gene therapy approaches for CF. For example, submucosal glands (SMGs) secrete mucous, airway surface fluid, and bactericidal proteins, and are a predominant site of CFTR-expressing cells in the lung. In contrast, cells in the surface airway epithelium show lower levels of CFTR expression (). The importance of SMGs in the pathoprogression of CF lung disease is still an open question. Hindering a firm answer to this question is a lack of animal models that mimic human CF airways disease at the molecular level and cellular levels.

Role of CFTR’s PDZ1-binding domain, NBF1 and Cl − conductance in inhibition of epithelial Na + channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits epithelial Na + channels (ENaC). Evidence has accumulated that both Cl − transport through CFTR Cl − channels and the first nucleotide binding domain (NBF1) of CFTR are crucial for inhibition of ENaC. A PDZ binding domain (PDZ-BD) at the C-terminal end links CFTR to scaffolding and cytoskeletal proteins, which have been suggested to play an important role in activation of CFTR and eventually inhibition of ENaC. We eliminated the PDZ-BD of CFTR and coexpressed Na +/H +-exchange regulator factors together with CFTR and ENaC. The results do not support a role of PDZ-BD in inhibition of ENaC by CFTR. However, inhibition of ENaC was closely linked to Cl − currents generated by CFTR and was observed in the presence of Cl −, I − or Br − but not gluconate. Therefore, functional NBF1 and Cl − transport are required for inhibition of ENaC in Xenopus oocytes, while the PDZ-BD is not essential.

Regulatory Interaction between the Cystic Fibrosis Transmembrane Conductance Regulator and HCO[formula omitted] Salvage Mechanisms in Model Systems and the Mouse Pancreatic Duct

The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO3- secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO3- secretion, the role of CFTR in HCO3- salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO3- salvage mechanism Na+/H+ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote DeltaF508 mice. Accordingly, luminal Na+-dependent and HOE694- sensitive recovery from an acid load was reduced by 60% in ducts of DeltaF508 mice. CFTR and NHE3 were co-immunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of DeltaF508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO3- homeostasis by regulating both pancreatic ductal HCO3- secretory and salvage mechanisms.

Role of CFTR in autosomal recessive polycystic kidney disease.

An extensive body of in vitro data implicates epithelial chloride secretion, mediated through cystic fibrosis transmembrane conductance regulator (CFTR) protein, in generating or maintaining fluid filled cysts in MDCK cells and in human autosomal dominant polycystic kidney disease (ADPKD). In contrast, few studies have addressed the pathophysiology of fluid secretion in cyst formation and enlargement in autosomal recessive polycystic kidney disease (ARPKD). Murine models of targeted disruptions or deletions of specific genes have created opportunities to examine the role of individual gene products in normal development and/or disease pathophysiology. The creation of a murine model of CF, which lacks functional CFTR protein, provides the opportunity to determine whether CFTR activity is required for renal cyst formation in vivo. Therefore, this study sought to determine whether renal cyst formation could be prevented by genetic complementation of the BPK murine model of ARPKD with the CFTR knockout mouse. The results of this study reveal that in animals that are homozygous for the cystic gene (bpk), the lack of functional CFTR protein on the apical surface of cystic epithelium does not provide protection against cyst growth and subsequent decline in renal function. Double mutant mice (bpk -/-; cftr -/-) developed massively enlarged kidneys and died, on average, 7 d earlier than cystic, non-CF mice (bpk -/-; cftr +/+/-). This suggests fundamental differences in the mechanisms of transtubular fluid secretion in animal models of ARPKD compared with ADPKD.

Generation and phenotype of cell lines derived from CF and non-CF mice that carry the H-2K(b)-tsA58 transgene.

Tracheal, renal, salivary, and pancreatic epithelial cells from cystic fibrosis [CF; cystic fibrosis transmembrane conductance regulator (CFTR) -/-] and non-CF mice that carry a temperature-sensitive SV40 large T antigen oncogene (ImmortoMouse) were isolated and maintained in culture under permissive conditions (33 degrees C with interferon-gamma). The resultant cell lines have been in culture for >1 year and 50 passages. Each of the eight cell lines form polarized epithelial barriers and exhibit regulated, electrogenic ion transport. The four non-CF cell lines (mTEC1, mCT1, mSEC1, and mPEC1) express cAMP-regulated Cl(-) permeability and cAMP-stimulated Cl(-) secretion. In contrast, the four CFTR -/- cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) each lack cAMP-stimulated Cl(-) secretory responses. Ca(2+)-activated Cl(-) secretion is retained in both CF and non-CF cell lines. Thus we have generated genetically well-matched epithelial cell lines from several tissues relevant to cystic fibrosis that either completely lack CFTR or express endogenous levels of CFTR. These cell lines should prove useful for studies of regulation of epithelial cell function and the role of CFTR in cell physiology.

Localization of Cystic Fibrosis Transmembrane Conductance Regulator in Epithelial Cells of Nasal Polyps and Postoperative Polypoid Mucosae

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel protein that plays an important role in electrolyte and water transport through the respiratory epithelial cells. In order to understand the possible role of CFTR in the pathogenesis of nasal polyps and postoperative polypoid mucosae, we aimed to characterize the localization of CFTR in the epithelia of nasal polyps and postoperative polypoid mucosae of subjects who did not manifest the phenotypic expression of cystic fibrosis. Immunohistochemical staining for CFTR, using monoclonal mouse anti-human CFTR, was performed on tissue sections of 4 normal turbinates and nasal polyps and postoperative polypoid mucosae from 10 patients who underwent endoscopic intranasal operations. CFTR showed a typical apical distribution in the normal turbinate mucosae whereas, in the nasal polyps, CFTR demonstrated a heterogenous pattern of localization comprising diffuse or scattered cytoplasmic labelling, very low to undetectable labelling, intense perinuclear staining and intermingled typical apical location. In postoperative polypoid mucosae, the pattern of CFTR localization was less heterogeneous than in the nasal polyp epithelial cells and showed a more prominent feature of diffuse cytoplasmic staining. These results suggest that an altered localization of the CFTR may have a role in the pathogenesis of nasal polyps and postoperative polypoid mucosa.

Unique presentations and chronic complications in adult cystic fibrosis: do they teach us anything about CFTR?

The increase in numbers of adults with cystic fibrosis (CF) has allowed us to identify previously unrecognized chronic complications of CF, as well as appreciate unique presentations of cystic fibrosis-related diseases. Do these chronic complications and unique presentations provide us with new insight into cystic fibrosis transmembrane conductance regulator (CFTR) function? Current data suggest that the 'chronic complications' reveal mainly the effect of a long-term absence of previously recognized CFTR functions. In contrast, the 'unique presentations' provide new insight into the role of CFTR in different tissues.

Evidence against the acidification hypothesis in cystic fibrosis.

The pleiotropic effects of cystic fibrosis (CF) result from the mislocalization or inactivity of an apical membrane chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR may also modulate intracellular chloride conductances and thus affect organelle pH. To test the role of CFTR in organelle pH regulation, we developed a model system to selectively perturb the pH of a subset of acidified compartments in polarized cells and determined the effects on various protein trafficking steps. We then tested whether these effects were observed in cells lacking wild-type CFTR and whether reintroduction of CFTR affected trafficking in these cells. Our model system involves adenovirus-mediated expression of the influenza virus M2 protein, an acid-activated ion channel. M2 expression selectively slows traffic through the trans-Golgi network (TGN) and apical endocytic compartments in polarized Madin-Darby canine kidney (MDCK) cells. Expression of M2 or treatment with other pH perturbants also slowed protein traffic in the CF cell line CFPAC, suggesting that the TGN in this cell line is normally acidified. Expression of functional CFTR had no effect on traffic and failed to rescue the effect of M2. Our results argue against a role for CFTR in the regulation of organelle pH and protein trafficking in epithelial cells.

Role of CFTR in airway disease.

Role of CFTR in Airway Disease. Physiol. Rev. 79, Suppl.: S215-S255, 1999. - Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), which accounts for the cAMP-regulated chloride conductance of airway epithelial cells. Lung disease is the chief cause of morbidity and mortality in CF patients. This review focuses on mechanisms whereby the deletion or impairment of CFTR chloride channel function produces lung disease. It examines the major themes of the channel hypothesis of CF, which involve impaired regulation of airway surface fluid volume or composition. Available evidence indicates that the effect of CFTR deletion alters physiological functions of both surface and submucosal gland epithelia. At the airway surface, deletion of CFTR causes hyperabsorption of sodium chloride and a reduction in the periciliary salt and water content, which impairs mucociliary clearance. In submucosal glands, loss of CFTR-mediated salt and water secretion compromises the clearance of mucins and a variety of defense substances onto the airway surface. Impaired mucociliary clearance, together with CFTR-related changes in the airway surface microenvironment, leads to a progressive cycle of infection, inflammation, and declining lung function. Here, we provide the details of this pathophysiological cascade in the hope that its understanding will promote the development of new therapies for CF.

Expression of CFTR in human and bovine thyroid epithelium.

The expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the thyroid has not been documented to date, although a role for CFTR in the thyroid follicular epithelium is suggested both clinically, by the occurrence of subclinical hypothyroidism in patients with cystic fibrosis (CF), and physiologically, by the presence of low-conductance, adenosine 3',5'-cyclic monophosphate-activated Cl channels in the follicular cells. Using reverse transcriptase-polymerase chain reaction with nested primers derived from exons 13 and 14 of the human CF gene, we have now documented the presence of CFTR mRNA in the human thyroid. Western blot analyses using six antibodies directed against different domains of human CFTR showed that a 165-kDa band was present in membrane extracts from bovine and human thyroid. This protein has the predicted size of mature CFTR and was not detected with preimmune serum or preadsorbed antiserum. By immunofluorescence and immunoperoxidase, CFTR was located in the follicular cells, with a diffuse, intracellular labeling pattern. Quantitative analysis revealed that 64% of the follicles were CFTR positive, but only 16% of the follicular cells were stained per follicle. The number of CFTR-positive cells was inversely proportional to the size of the follicle. These results 1) demonstrate the expression of CFTR at the mRNA and protein levels in human and bovine thyroid follicular cells and 2) suggest that CFTR expression could be instrumental in follicular enlargement.

A role for CFTR in human autosomal dominant polycystic kidney disease.

Human autosomal dominant polycystic kidney disease (ADPKD) is the most common lethal dominant hereditary disorder characterized by enormous renal enlargement and the development of multiple cysts originating from nephrons. We investigated the pathogenesis of cyst formation in ADPKD by using patch-clamp and immunocytochemical techniques. Adenosine 3',5'-cyclic monophosphate-activated Cl- currents are present in primary cultures of ADPKD cells and have characteristics such as a linear current-voltage relation, insensitivity to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, sensitivity to glibenclamide and diphenylamine carboxylic acid, and an anion selectivity sequence of Br- > Cl- > I- > glutamate, all of which are identical to cystic fibrosis transmembrane conductance regulator (CFTR). With the use of CFTR antibodies raised against the regulatory and first nucleotide-binding domains, CFTR was detected in primary cultures of ADPKD cells. Similar results were obtained in vivo in cyst-lining epithelial cells in ADPKD kidneys, where staining was seen associated with the apical membrane regions. These data indicate that the CFTR Cl- channel exists in apical membranes of ADPKD cells and may play an important role in cyst formation or enlargement.

CFTR gene transfer corrects defective glycoconjugate secretion in human CF epithelial tracheal cells

We demonstrate that in immortalized normal human tracheal epithelial cells (NT-1 and 56FHTE8o-) 14C-labeled glycoconjugate secretion may be regulated independently by agonists of the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways. In contrast, in immortalized cystic fibrosis (CF) human tracheal epithelial cells (CFT-1 and CFT-2), regulation is defective for agonists specific for the PKA but not for the PKC pathway. To characterize the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in regulated glycoconjugate secretion, we examined the effect of adenovirus-mediated gene transfer of CFTR to CF and control cells. Forty-eight hours after infection, at a multiplicity of infection of 50 plaque-forming units per cell, high levels of CFTR mRNA were detected by reverse transcription-polymerase chain reaction, and de novo synthesis of CFTR protein was demonstrated by immunoblotting. Gene transfer to CF cells restored defective adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretion not only of chloride but also of glycoconjugates. Taken together, these results argue for a role for CFTR in cAMP-mediated glycoconjugate secretion.

Role of CFTR in chloride secretion across human tracheal epithelium.

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.

Coupled secretion of chloride and mucus in skin of Xenopus laevis: possible role for CFTR

We used the isolated skin of Xenopus laevis to investigate the relationship between the secretion of salt, water, and mucus by submucosal glands expressing the cystic fibrosis transmembrane conductance regulator (CFTR). In situ hybridization and immunofluorescence provided evidence for specific expression of CFTR in the mucus-secreting cells of the subepidermal glands. Stimulation of isolated sheets of skin with 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate produced active Cl secretion and a marked increase in tissue conductance that was correlated with mucous cell degranulation and the distention of the glandular ducts. This coordinated increase in active secretion of salt and mucus was abolished by pretreatment of skins with bumetanide or by removing Cl from the bathing solutions. These results provide evidence for an intimate coupling between electrolyte transport and mucus secretion that may illuminate the pathophysiology of mucus-producing glands in cystic fibrosis lung disease.

Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is found at the apical region of exocrine epithelial cells, both at the cell surface and in an apically localized intracellular compartment. To determine if this internal pool was due to endocytosis, a technique was developed that allows the rate of CFTR internalization from the cell surface to be monitored. A two-step periodate/hydrazide biotinylation procedure was used to derivatize cell surface glycoconjugates. Because both of these steps are required for derivatization and are conducted at 4 degrees C, the inclusion of a 37 degrees C incubation between the treatments resulted in an assay for the internalization of cell surface glycoconjugates. CFTR was found to be targeted to a rapidly recycling endocytic pathway, as approximately 50% of cell surface CFTR was internalized within minutes and unavailable for biotinylation. In contrast, the major glycoproteins of the apical surface were not significantly endocytosed during even longer incubations at 37 degrees C. Elevating cAMP levels either by forskolin or cAMP analogs, which has been shown to activate CFTR chloride channel activity, inhibited CFTR internalization. However, cAMP did not affect the internalization of G551D CFTR, a naturally occurring Gly-551-->Asp mutant that is expressed at the cell surface but lacks normal ion-channel function. In addition, the inhibition by cAMP of CFTR was not observed when cells were depleted of cellular chloride. The presence of CFTR in epithelial cells had previously been shown to confer a cAMP-mediated inhibition on the rate of fluid-phase endocytosis. This effect was not seen in chloride-depleted cells, suggesting that CFTR's ion-channel function and localization to incipient endosomes may be responsible for the observed inhibition. The finding that CFTR is targeted to the endocytic pathway may provide insight into the role of CFTR in normal exocrine function. In addition, these findings suggest that the expression of a regulated ion channel in a membranous subcellular compartment provides a mechanism by which a cell can regulate vesicular trafficking through that compartment.

Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells

Background: Liver dysfunction is a common manifestation of cystic fibrosis (CF), a disease caused by mutations affecting the CF transmembrane conductance regulator (CFTR). The aim of this study was to examine the distribution and role of CFTR in liver. Methods: CFTR messenger RNA was detected in cryosections of human liver by in situ hybridization. CFTR immunoreactivity was detected using antibodies raised against two CFTR peptides. Results: The predominant site of CFTR messenger RNA and immunoreactivity in liver is the intrahepatic bile duct. CFTR is not detected in hepatocytes of normal liver or in livers exhibiting bile duct proliferation. Within bile duct cells, CFTR is localized at or near the apical plasma membrane. Conclusions: The apical localization of CFTR in bile duct cells suggests a model explaining how the CFTR-associated Cl− channel contributes to normal biliary secretion. This model suggests that if CFTR expression could be promoted in intrahepatic duct cells by somatic gene therapy, this might prevent the occurrence of liver disease in CF.