Wild-type Cystic Fibrosis Transmembrane Conductance Research Articles

Hypoxia-induced cystic fibrosis transmembrane conductance regulator dysfunction is a universal mechanism underlying reduced mucociliary transport in sinusitis.

Hypoxia due to sinus obstruction is a major pathogenic mechanism leading to sinusitis. The objective of the current study is to define the electrophysiologic characteristics of hypoxia in vitro and in vivo. Cystic fibrosis bronchoepithelial cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and human sinonasal epithelial cells were exposed to 1% or atmospheric O2 for 24h. Time-dependent production of cytoplasmic free radicals was measured. Cells were subjected to Ussing chamber and patch clamp technique where CFTR currents were recorded in whole-cell and cell-attached mode for single channel studies. Indices of mucociliary transport (MCT) were measured using micro-optical coherence tomography. In a rabbit hypoxic maxillary sinus model, tissue oxygenation, relative mRNA expression of HIF-1α, pH, sinus potential difference (SPD), and MCT were determined. Ussing chamber (p<0.05), whole-cell (p<0.001), and single channel patch-clamp (p<0.0001) showed significant inhibition of Cl- currents in hypoxic cells. Cytoplasmic free radicals showed time-dependent elevation peaking at 4h (p<0.0001). Airway surface liquid (p<0.0001), periciliary liquid (p<0.001), and MCT (p<0.01) were diminished. Co-incubation with the free radical scavenger glutathione negated the impact of hypoxia on single channel currents and MCT markers. In sinusitis rabbits, mucosa exhibited low tissue oxygenation (p<0.0001), increased HIF1α mRNA (p<0.05), reduced pH (p<0.01), and decreased MCT (p<0.001). SPD measurements demonstrated markedly diminished transepithelial Cl- transport (p<0.0001). Hypoxia induces severe CFTR dysfunction via free radical production causing reduced MCT in vitro and in vivo. Improved oxygenation is critical to reducing the impact of persistent mucociliary dysfunction.

EPS3.01 Analysis of synthetic derivatives with reduced lipophilicity as candidate potentiators towards better treatment alternatives for cystic fibrosis

Objectives: KALYDECO (ivacaftor/VX-770), a potentiator that enhances channel gating of wild-type cystic fibrosis transmembrane conductance regulator (CFTR) protein and certain CFTR gating mutants was first approved to treat Cystic Fibrosis (CF). Thereafter, Orkambi, a combination of ivacaftor and the trafficking corrector lumacaftor was approved for patients with homozygous F508del mutation. Unfortunately, ORKAMBI mediates modest and variable clinical responses which relates to a negative impact of VX-770 on the metabolic stability of F508del-CFTR. VX-770 was also shown to exert a negative effect on the stability of other membrane solute carriers. The destabilizing effect of VX-770 also correlated with its predicted lipophilicity, suggesting the need for discovery and optimization of efficacious potentiators to maximize the clinical benefit in CF.

ENaC, iNOS, mucins, and wound healing in cystic fibrosis airway epithelial and submucosal cells

We compared airway epithelial cell models relevant for cystic fibrosis (CF): 16HBE cells with endogenous wild-type cystic fibrosis transmembrane conductance regulator (CFTR), CFBE cells with mutated ΔF508-CFTR, corrected CFBE cells overexpressing CFTR, CFSME (CF submucosal) and Calu-3 (non-CF submucosal) cells with respect to the epithelial sodium channel (ENaC), inducible NO synthase (iNOS) and mucins (MUC) (studied by quantitative Real-Time-Polymerase Chain Reaction, qRT-PCR and Western blot), and wound healing. CFBE cells had significantly more expression of β- and γ-ENaC mRNA and of β-ENaC protein than 16HBE cells. Compared to corrected CFBE cells, CFBE cells had increased mRNA expression of all ENaC subunits and β-ENaC protein. For ENaC, the CFSME/Calu-3 mRNA ratio was very low and contradictory to the ENaC upregulation in CF cells. CFBE cells showed decreased expression of iNOS at both mRNA and protein levels compared to 16HBE cells and only at the mRNA level compared to corrected CFBE cells. CFSME cells showed expression of iNOS whereas Calu-3 cells did not. Higher expression of MUC2 and MUC5B was found in corrected CFBE cells compared to CFBE cells. Wound healing in CFBE cells was delayed compared to corrected CFBE cells, but not to 16HBE cells, and in CFSME cells compared to Calu-3 cells. Our data suggest CFSME as an inappropriate CF cell model for Calu-3 cells, and provide partial support for the theory that the differences (in ENaC, iNOS and wound healing) between these cell lines are associated to the presence of CFTR in the bronchial airway epithelial cells.

Impaired airway epithelial barrier function in cystic fibrosis related diabetes

Cystic Fibrosis (CF)‐Related Diabetes (CFRD) impacts ~50% of adult CF patients. CFRD is associated with abnormally high airway glucose and an accelerated decline in pulmonary function, indicating a failure in the airway epithelial barrier to regulate glucose permeability. Thus, we examined tight junctions of airway cells expressing wild type CF Transmembrane Conductance Regulator (CFTR) to cells expressing mutant ΔF508‐CFTR. Increasing basolateral glucose from 100 to 450 mg/dL compromised tight junctions of ΔF508‐CFTR cells by ~25% vs. control cells which were significantly less affected. Impaired barrier function was associated with decreased expression of claudin‐4 and displacement of junctional ZO‐1. Using a dual‐luciferase‐ based ER stress reporter assay, cells expressing ΔF508‐ CFTR exhibited higher levels of ER stress than control cells. ER stress was further induced upon exposure to high glucose medium. Thus, ER stress amplified by expression of ΔF508‐CFTR impairs the airway glucose barrier and may predispose the lungs of CF patients to the detrimental consequences of systemic diabetes. Support: Emory+Children's Center for CF Research, Emory URC, CFF, NIH‐NIAAA, NIH‐NHLBI.

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.

Gene and cell therapy for cystic fibrosis: From bench to bedside

Clinical trials in cystic fibrosis (CF) patients established proof-of-principle for transfer of the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells. However, the limited efficacy of gene transfer vectors as well as extra- and intracellular barriers have prevented the development of a gene therapy-based treatment for CF. Here, we review the use of new viral and nonviral gene therapy vectors, as well as human artificial chromosomes, to overcome barriers to successful CFTR expression. Pre-clinical studies will surely benefit from novel animal models, such as CF pigs and ferrets. Prenatal gene therapy is a potential alternative to gene transfer to fully developed lungs. However, unresolved issues, including the possibility of adverse effects on pre- and postnatal development, the risk of initiating oncogenic or degenerative processes and germ line transmission require further investigation. Finally, we discuss the therapeutic potential of stem cells for CF lung disease.

Sensitivity of Chloride Efflux vs. Transepithelial Measurements in Mixed CF and Normal Airway Epithelial Cell Populations

Background/Aims: While the Cl^- efflux assays are relatively straightforward, their ability to assess the efficacy of phenotypic correction in cystic fibrosis (CF) tissue or cells may be limited. Accurate assessment of therapeutic efficacy, i.e., correlating wild type CF transmembrane conductance regulator (CFTR) levels with phenotypic correction in tissue or individual cells, requires a sensitive assay. Methods: Radioactive chloride (³⁶Cl) efflux was compared to Ussing chamber analysis for measuring cAMP-dependent Cl^- transport in mixtures of human normal (16HBE14o-) and cystic fibrosis (CF) (CFTE29o- or CFBE41o-, respectively) airway epithelial cells. Cell mixtures with decreasing amounts of 16HBE14o- cells were evaluated. Results: Efflux and Ussing chamber studies on mixed populations of normal and CF airway epithelial cells showed that, as the number of CF cells within the population was progressively increased, the cAMP-dependent Cl^- decreased. The ³⁶Cl efflux assay was effective for measuring Cl^- transport when ≥ 25% of the cells were normal. If < 25% of the cells were phenotypically wild-type (wt), the ³⁶Cl efflux assay was no longer reliable. Polarized CFBE41o- cells, also homozygous for the ΔF508 mutation, were used in the Ussing chamber studies. Ussing analysis detected cAMP-dependent Cl^- currents in mixtures with ≥1% wild-type cells indicating that Ussing analysis is more sensitive than ³⁶Cl efflux analysis for detection of functional CFTR. Conclusions: Assessment of CFTR function by Ussing analysis is more sensitive than ³⁶Cl efflux analysis. Ussing analysis indicates that cell mixtures containing 10% 16HBE14o- cells showed 40-50% of normal cAMP-dependent Cl^- transport that drops off exponentially between 10-1% wild-type cells.

Nonsteroidal anti-inflammatory drugs upregulate function of wild-type and mutant CFTR

Small-scale clinical trials show that treatment of cystic fibrosis (CF) patients with ibuprofen, a nonsteroidal anti-inflammatory drug, improves the symptoms of CF and slows down the decline of lung function. Paradoxically, ibuprofen inhibits ligand-stimulated CF transmembrance conductance regulator (CFTR) activity. The aim of the present study was to investigate the effects of ibuprofen on CFTR function under different conditions. Patch-clamp recordings were performed in two lines of human airway epithelial cells: IB3-8-3-7 cells, which express wild-type CFTR; and IB3-1 cells, which express the variant CFTR with deletion of phenylalanine 580 (DeltaF580CFTR). Addition of ibuprofen to the extracellular solution caused a rapid inhibition of CFTR activity in IB3-8-3-7 cells in the presence of a high intracellular concentration of cAMP, whereas ibuprofen enhanced the CFTR conductance at low levels of cAMP. Introducing ibuprofen into the interior of cells occluded the enhancing effect of ibuprofen. Notably, the variant CFTR-mediated conductance was detected in IB3-1 cells treated with myoinositol and was enhanced by ibuprofen at endogenous levels of cAMP. In summary, nonsteroidal anti-inflammatory drugs increase the function of both wild-type cystic fibrosis transmembrane conductance regulator and the phenylalanine 580 deletion in cultured human airway epithelial cells at endogenous levels of cAMP.

Determination of the Functional Unit of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel: ONE POLYPEPTIDE FORMS ONE PORE

The magnitudes and distributions of subconductance states were studied in chloride channels formed by the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and in CFTRs bearing amino acid substitutions in transmembrane segment 6. Within an open burst, it was possible to distinguish three distinct conductance states referred to as the full conductance, subconductance 1, and subconductance 2 states. Amino acid substitutions in transmembrane segment 6 altered the duration and probability of occurrence of these subconductance states but did not greatly alter their relative amplitudes. Results from real time measurements indicated that covalent modification of single R334C-CFTR channels by [2-(trimethylammonium)ethyl]methanethiosulfonate resulted in the simultaneous modification of all three conductance levels in what appeared to be a single step, without changing the proportion of time spent in each state. This behavior suggests that at least a portion of the conduction path is common to all three conducting states. The time course for the modification of R334C-CFTR, measured in outside-out macropatches using a rapid perfusion system, was also consistent with a single modification step as if each pore contained only a single copy of the cysteine at position 334. These results are consistent with a model for the CFTR conduction pathway in which a single anion-conducting pore is formed by a single CFTR polypeptide.

Hypoxia Increases Corneal Cell Expression of CFTR Leading to IncreasedPseudomonas aeruginosaBinding, Internalization, and Initiation of Inflammation

To investigate the effect of hypoxia-induced molecular responses of corneal epithelial cells on the surface of rabbit and human corneas and corneal cells in culture on interactions with Pseudomonas aeruginosa that may underlie increased susceptibility to keratitis. Organ cultures of rabbit and human corneal tissue, primary rabbit and human corneal cells, and transformed human corneal cells from a patient with cystic fibrosis and the same cell line corrected for expression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR), the cellular receptor for P. aeruginosa, were exposed to hypoxic conditions for 24 to 72 hours. Changes in binding and internalization of P. aeruginosa were measured using cellular association and gentamicin-exclusion assays, and expression of CFTR and activation of NF-kappaB in response to hypoxia were determined by confocal laser microscopy and quantitative measurements of NF-kappaB activation. Hypoxia induced in a time- and oxygen-concentration-dependent manner increased association and internalization of clinical isolates of P. aeruginosa in all cells tested. Hypoxia increased CFTR expression and NF-kappaB nuclear translocation in rabbit and human cells with wild-type CFTR. Corneal cells lacking CFTR had reduced NF-kappaB activation in response to hypoxia. Hypoxia did not affect the increase in corneal cell CFTR levels or NF-kappaB activation after P. aeruginosa infection. Hypoxic conditions on the cornea exacerbate the binding and internalization of P. aeruginosa due to increased levels of CFTR expression and also induce basal NF-kappaB activation. Both of these responses probably exacerbate the effects of P. aeruginosa infection by allowing lower infectious doses of bacteria to induce disease and promote destructive inflammatory responses.

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry.

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.

Identification of a region of strong discrimination in the pore of CFTR.

The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.

Pharmacological treatment of the biochemical defect in cystic fibrosis airways.

The understanding of the biochemical defect in cystic fibrosis (CF) has advanced considerably since discovery of the CF gene in 1989 and characterization of its product. Studies showing that the abnormality in chloride flux could be corrected by transfection of wild-type cystic fibrosis transmembrane conductance regulator (CFTR) complimentary deoxyribonucleic acid (cDNA) have led to gene therapy trials on both sides of the Atlantic. However, gene therapy as a treatment for CF has yet to be realized. Pharmacological manipulation of the biochemical defect may provide an alternative or complementary approach to treatment. This review will discuss pharmacological agents in development which could correct the abnormal ion movement. The mechanisms of action of these pharmacological agents can be divided broadly into drugs which affect the most common CF mutation, deltaF508, which increase trafficking of the mutant CF protein to the apical membrane; drugs which increase chloride secretion; and drugs which reduce sodium reabsorption across the apical membrane. Treatment options for cystic fibrosis have developed rapidly since discovery of the cystic fibrosis gene over a decade ago. The targeting of specific therapies for particular cystic fibrosis genotypes and the use of combination treatments of chloride channel openers with sodium channel blockers are likely to be key advances in the next decade.

The phorbol ester PMA and cyclic AMP activate different Cl − and HCO 3− fluxes in C127 cells expressing CFTR

In mouse mammary epithelial C127 cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR), chloride efflux, measured with the Cl −-sensitive dye 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ), was stimulated by activation of protein kinase A with cyclic AMP elevating agents forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) and, to a less extent, by activation of protein kinase C with the phorbol 12-myristate 13-acetate (PMA). Conversely, bicarbonate influx, determined by intracellular alkalinization of cells incubated with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluoresceintetraacetoxymethyl ester (BCECF-AM), was stimulated by cyclic AMP elevation, but not by PMA. Patch clamp analysis revealed that PMA activated a Cl − current with the typical biophysical characteristics of swelling-activated current and not of CFTR.

ENHANCEMENT OF LIPOSOME-MEDIATED GENE TRANSFER TO HUMAN AIRWAY EPITHELIAL CELLS BY REPLICATION-DEFICIENT ADENOVIRUS

We hypothesized that replication-deficient adenovirus (Ad), when complexed with plasmid DNA (pl) and cationic liposomes (L), would enhance liposome-mediated gene transfer in cultured human airway epithelial cells. Pl/L/Ad complexes were formed using charge-charge interactions. A gel electrophoresis retardation assay showed plasmid DNA to be associated with the virus in a high-molecular weight, low-mobility complex, the diameter of which was 300 to 350 nm. Compared to pl/L alone, pl/L/Ad enhanced luciferase expression on average by 1 log-fold in human airway epithelial cells which express either mutant or wild-type cystic fibrosis transmembrane conductance regulator (CFTR). Transgene expression was sustained at high levels for up to 7 days following transfection with pl/L/Ad. Using a heat-stable alkaline phosphatase reporter gene, we showed that a larger fraction of cells was transfected by pl/L/Ad compared to pl/L. Finally, cells were exposed to Ad for 0 to 24 hours prior to pl/L or exposed to pl/L prior to Ad. We found that enhancement was significantly greater using pl/L/Ad compared to the simultaneous addition of Ad with the pl/L complexes. In addition, when pl/L was added 4 to 24 hours prior to Ad, some enhancement was found, suggesting that plasmid DNA remained in a compartment in the cell for several hours and became available for transcription with the addition of Ad. When Ad was added prior to pl/L, enhancement was found suggesting that the effect of the virus on cell membranes may persist for up to 24 hours. We conclude that the tripartite pl/L/Ad complex significantly enhances liposome-mediated transgene expression for a prolonged period of time in human bronchial epithelial cells.

Nitric oxide inhibits heterologous CFTR expression in polarized epithelial cells.

Nitric oxide (. NO) has been implicated in a wide range of autocrine and paracrine signaling mechanisms. Herein, we assessed the role of exogenous. NO in the modulation of heterologous gene expression in polarized kidney epithelial cells (LLC-PK(1)) that were stably transduced with a cDNA encoding human wild-type cystic fibrosis transmembrane conductance regulator (CFTR) under the control of a heavy metal-sensitive metallothionein promoter (LLC-PK(1)-WTCFTR). Exposure of these cells to 125 microM DETA NONOate at 37 degrees C for 24 h (a chemical. NO donor) diminished Zn(2+)-induced and uninduced CFTR protein levels by 43.3 +/- 5.1 and 34.4 +/- 17.1% from their corresponding control values, respectively. These changes did not occur if red blood cells, effective scavengers of. NO, were added to the medium. Exposure to. NO did not alter lactate dehydrogenase release in the medium or the extent of apoptosis. Coculturing LLC-PK(1)-WTCFTR cells with murine fibroblasts that were stably transduced with the human inducible. NO synthase cDNA gene also inhibited CFTR protein expression in a manner that was antagonized by 1 mM N(G)-monomethyl-L-arginine in the medium. Pretreatment of LLC-PK(1)-WTCFTR with ODQ, an inhibitor of guanylyl cyclase, did not affect the ability of. NO to inhibit heterologous CFTR expression; furthermore, 8-bromo-cGMP had no effect on heterologous CFTR expression. These data indicate that. NO impairs the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms.

Cystic fibrosis transmembrane conductance regulator activation by cAMP-independent mechanisms.

Recent studies have demonstrated that several compounds with diverse structures can activate wild-type cystic fibrosis transmembrane conductance regulator (CFTR) by non-receptor-mediated mechanisms. Some of these compounds have been shown to enhance cAMP-dependent activation of DeltaF508-CFTR. This study was undertaken to compare the mechanisms by which genistein, IBMX, milrinone, 8-cyclopentyl-1, 3-dipropylxanthine (CPX), the benzimidazolone NS004, and calyculin A increase CFTR activity. Our studies demonstrate that, in transfected NIH-3T3 cells, maximal enhancements of forskolin-dependent DeltaF508-CFTR activity are greatest with genistein, IBMX, and NS004. Milrinone, genistein, CPX, NS004, and calyculin A do not increase cellular cAMP. Because forskolin and calyculin A increase in vivo phosphorylation of cAMP binding response element (CREB), the inability of milrinone, genistein, CPX, and NS004 to increase CREB phosphorylation suggests that they do not stimulate protein kinase A or inhibit phosphatase activity. Our data suggest that the mechanisms by which genistein and NS004 activate CFTR differ. We also demonstrate that, in NIH-3T3 cells, IBMX-dependent enhancement of cAMP-dependent CFTR activity is not due to an increase in cellular cAMP and may involve a mechanism like that of genistein.

The role of exocytosis in the activation of the chloride conductance in Chinese hamster ovary cells (CHO) stably expressing CFTR.

The aim of this study was to examine the question of whether activation of wt-CFTR (wild-type cystic fibrosis transmembrane conductance regulator) by cAMP and the opening of a Cl- conductance is paralleled by exocytosis and corresponding increases in membrane capacitance. To this end three types of Chinese hamster ovary (CHO) cells were examined: a control group of CHO cells; a group of CHO cells stably expressing wt-CFTR at high levels (also called BQ2-CHO); and a group of CHO cells stably expressing the frequent mutation DeltaF508-CFTR. Whole-cell patch-clamp studies were performed to measure the membrane voltage (Vm), the membrane conductance (Gm) and the membrane capacitance (Cm). Cm was assessed by a two-frequency lock-in amplifier method. Forskolin (Fsk, 0.1 micromol/l) and isobutylmethylxanthine (IBMX, 0.1 mmol/l) were used to increase cytosolic cAMP. It is shown that Fsk and IBMX had no effect on Vm and Gm in control CHO and DeltaF508-CFTR-CHO cells. Fsk and IBMX depolarized wt-CFTR-expressing CHO cells significantly (from -40 +/- 1.5 to -32 +/- 1.6 mV, n = 41) and enhanced Gm strongly from 5.0 +/- 0.9 to 36 +/- 3.9 nS (n = 65). The conductance increase was mostly for Cl-, because under stimulated conditions a reduction in bath Cl- concentration depolarized these cells further and significantly from -26 +/- 1.8 to -10 +/- 1.2 mV (n = 16). This conductance had the characteristic wt-CFTR selectivity of Br- > Cl- > I- (n = 16). Despite this large increase in the Fsk- and IBMX-induced conductance Cm was not altered significantly (15.5 versus 15.7 pF, n = 50). These data indicate that stable overexpression of wt-CFTR but not of DeltaF508-CFTR in CHO cells induces a cAMP-activated Cl- conductance. The activation of this large conductance obviously proceeds with little if any exocytosis.

Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers

The involvement of tyrosine phosphorylation in the regulation of epithelial cell Cl- secretion is unknown. Therefore, the purpose of these studies was to determine if tyrosine kinase activation was involved in the regulation of Cl- secretion, using the tyrosine kinase inhibitors, genistein and tyrphostin 47, and human intestinal epithelial cells (T84 cells) as an intestinal Cl- secretory model. Genistein rapidly but reversibly stimulated sustained apical Cl- secretion in monolayers of T84 cells without increasing intracellular cyclic nucleotides or Ca2+ levels. Tyrphostin 47 also stimulated Cl- secretion in T84 monolayers, although it was short-lived. Transfection experiments in 3T3 fibroblasts and IEC-6 intestinal cells utilizing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) showed that genistein and tyrphostin 47 stimulated 125I efflux only in CFTR-transfected cells and not in CFTR-negative cells. Thus genistein- and tyrphostin 47-stimulated Cl- secretion involved CFTR. Genistein also acted synergistically with the Ca(2+)- and protein kinase C-dependent acetylcholine analogue, carbachol, to stimulate Cl- secretion in T84 monolayers. However, the Cl- secretory response to saturating concentrations of the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin, or the guanosine 3',5'-cyclic monophosphate (cGMP) agonist, Escherichia coli heat-stable enterotoxin, was not further enhanced by genistein. Although the mechanism of activation of Cl- secretion is unclear, these data suggest that tyrosine kinase activity limits basal Cl- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical membrane Cl- secretion, most likely through activation of the CFTR-Cl- channel. Moreover, genistein does not itself act through cAMP or cGMP elevation but appears to share a common Cl- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory responses to a Ca(2+)- and protein kinase C-dependent agonist.

Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins.

Mutant (delta F508) and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) were synthesized initially as an approximately 140-kDa core-glycosylated precursor, which, in the case of wild-type CFTR, was chased to an approximately 160 kDa form bearing complex oligosaccharides. Mutant CFTR disappeared from the detergent-soluble cell extract with rapid (t1/2 = 27 min) kinetics. Only approximately 25% of the initially synthesized wild-type 140-kDa CFTR precursor was detected as mature protein; the remaining approximately 75% decayed with kinetics (t1/2 = 33 min) indistinguishable from those of the mutant. Rapid degradation kinetics and inefficient processing of wild-type CFTR were also observed in the colonic carcinoma lines HT29 and T84 and in stably transfected C127 cells, which express 5-50 times lower levels of CFTR. These results suggest that inefficient processing and rapid degradation of wild-type CFTR precursor are an intrinsic property of CFTR in these diverse cell types and are not an artifact of overexpression. Degradation of wild-type and mutant 140-kDa CFTR began without significant lag following synthesis. These data suggest that wild-type and delta F508 CFTR differ in the efficiency of folding of the core-glycosylated primary translation product.