Regulation of Cl-/ HCO3- exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells

A central function of cystic fibrosis transmembrane conductance regulator (CFTR)-expressing tissues is the secretion of fluid containing 100-140 mM HCO3-. High levels of HCO3- maintain secreted proteins such as mucins (all tissues) and digestive enzymes (pancreas) in a soluble and/or inactive state. HCO3- secretion is impaired in CF in all CFTR-expressing, HCO3--secreting tissues examined. The mechanism responsible for this critical problem in CF is unknown. Since a major component of HCO3- secretion in CFTR-expressing cells is mediated by the action of a Cl-/HCO3- exchanger (AE), in the present work we examined the regulation of AE activity by CFTR. In NIH 3T3 cells stably transfected with wild type CFTR and in HEK 293 cells expressing WT and several mutant CFTR, activation of CFTR by cAMP stimulated AE activity. Pharmacological and mutagenesis studies indicated that expression of CFTR in the plasma membrane, but not the Cl- conductive function of CFTR was required for activation of AE. Furthermore, mutations in NBD2 altered regulation of AE activity by CFTR independent of their effect on Cl- channel activity. At very high expression levels CFTR modified the sensitivity of AE to 4,4'-diisothiocyanatostilbene-2, 2'-disulfonate. The novel finding of regulation of Cl-/HCO3- exchange by CFTR reported here may have important physiological implications and explain, at least in part, the impaired HCO3- secretion in CF.     

Antimitotic and tubulin-interacting properties of vinflunine, a novel fluorinated Vinca alkaloid

Cystic fibrosis (CF) lung disease has been linked to multiple primary defects in airway epithelia caused by a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) gene. These defects include altered Cl- and Na+ permeability as well as intracellular defects in glycoprotein processing. This apparent diversity in CFTR function is reflected in the complex patterning of CFTR expression in airway epithelia. Such complexities present challenges in the design of CF gene therapies that are capable of reconstituting the endogenous patterns of CFTR gene expression in appropriate target cells. Using a human bronchial xenograft model of the CF airway, we have evaluated the efficacy of recombinant adenoviral and cationic liposome-mediated gene transfer to correct Cl- permeability and mucous sulfation defects found in CF lung disease. Results from these studies demonstrated a clear vector-specific complementation profile for these two defects that was dependent on the type of cell transduced and the level of transgene expression. Single-dose administration of recombinant adenovirus effectively transduced high levels of CFTR transgene expression in 11 +/- 1% of epithelial cells and was capable of correcting cAMP-induced changes in Cl- permeability to 91 +/- 14% that seen in non-CF airways. However, this level of transgene expression was incapable of reversing defects in mucous sulfation due to the lack of efficient targeting to goblet cells. In contrast, cationic liposome-mediated delivery of CFTR encoding plasmids to CF airways achieved extremely low levels of transgene expression with insignificant correction (7.4 +/- 2.4%) of cAMP-induced Cl- permeability. This low level of transgene expression, however, efficiently reduced mucous sulfation to levels seen in non-CF airways. Differences in the complementation profiles of these two vectors in correcting Cl- permeability and mucous sulfation defects mirror the ability of recombinant adenovirus and liposomes to reconstitute only certain features of the endogenous distribution and abundance of CFTR protein expression. Such findings suggest that the level of intracellular CFTR required to facilitate proper glycoprotein processing may be much lower than that needed to mediate bulk Cl- flow across the airway epithelium. In summary, these data present the first example by which two different vector systems can efficiently complement independent primary defects associated with a single dysfunctional gene.     

Understanding how cystic fibrosis mutations cause a loss of Cl- channel function

Defective epithelial Cl- secretion is the hallmark of the lethal genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a regulated Cl- channel. Since the identification of the single gene encoding CFTR, several hundred disease-causing mutations, associated with a wide variety of clinical phenotypes, have been reported. To understand the relationship between genotype and clinical phenotype, researchers have investigated how mutations in CFTR disrupt its function. Here, we review the recent progress in understanding how CF-associated mutations in CFTR produce defective Cl- channels, and discuss the implications of this knowledge for the development of therapy for CF.     

Role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator

The anion-selective channel CFTR (cystic fibrosis transmembrane conductance regulator), whose dysfunction is responsible for the onset of cystic fibrosis, is regulated by cAMP through the activation of protein kinase A (PKA). The nature of this activation process is unknown. In the present study, patch-clamp techniques were applied to both mouse mammary adenocarcinoma cells expressing human epithelial CFTR (CFTR cells) and cultured neonatal rat ventricular myocytes (NRVM), to determine whether CFTR is modulated by the actin cytoskeleton, and whether the actin cytoskeleton may be implicated in the cAMP-stimulated activation of the channel protein. Acute changes in the actin cytoskeleton by addition of cytochalasin D (CD) activated whole-cell currents in CFTR cells and NRVM. Addition of actin to excised, inside-out patches also activated CFTR. A functional characterization of CFTR in either cell type included cAMP-induced, linear whole-cell and single-channel currents in symmetrical Cl-, permeability to ATP, and inhibition by either diphenylamine-carboxylate (DPC) or a monoclonal antibody raised against CFTR. Incubation of CFTR cells and NRVM with CD for over 6 h prevented CFTR activation either by the cAMP pathway under whole-cell conditions or by PKA under excised inside-out conditions. Thus a complete derangement of the actin cytoskeleton prevents the cAMP-dependent activation of CFTR. CFTR activation, however, was restored by subsequent addition of actin. In summary, changes in actin filament organization modulate CFTR channel activity by a mechanism entailing a direct interaction between actin filaments and CFTR.     

Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl-) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl- secretion by stimulating CFTR Cl- channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patch-clamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl- secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl- secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.     

Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl- that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl- conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl- conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl > Br; R347P, Cl > Br; R347E, Br > Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl- binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl-. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.     

Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.     

Cystic fibrosis transmembrane regulator-independent release of ATP. Its implications for the regulation of P2Y2 receptors in airway epithelia

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel that is defective in CF cells. It has been hypothesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-independent Ca2+-activated Cl- conductance is regulated by the P2Y2 receptor. Thus, ATP may function as an autocrine signaling factor promoting Cl- secretion in normal but not CF epithelia if ATP release is defective. We have tested for CFTR-dependent ATP release using four independent detection systems. First, a luciferase assay detected no differences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A marked accumulation of extracellular ATP resulted from mechanical stimulation effected by a medium displacement. Second, high pressure liquid chromatography analysis of 3H-labeled species released from [3H]adenine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells. Mechanical stimulation of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-33P]dADP, equivalent formation of [33P]dATP was observed in the media of control and cyclic AMP-stimulated HNE cells and nasal epithelial cells from wild-type and CF mice. Mechanically stimulated [33P]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y2 receptor were used as a reporter system for detection of ATP via P2Y2 receptor-promoted formation of [3H]inositol phosphates. Basal [3H]inositol phosphate accumulation was of the same magnitude in control and CFTR-transduced cells, and no change was observed following addition of forskolin and isoproterenol. In both cell types, mechanical stimulation resulted in hexokinase-attenuable [3H]inositol phosphate formation. In summary, our data suggest that ATP release may be triggered by mechanical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP.     

Cellular heterogeneity of CFTR expression and function in the lung: implications for gene therapy of cystic fibrosis

Cystic fibrosis (CF) has become a paradigm disorder for the clinical testing of gene therapies in the treatment of inherited disease. In recent years, efforts directed at gene therapy of CF have concentrated on improving gene delivery systems to the airway. Surrogate endpoints for complementation of CFTR dysfunction in the lung have been primarily dependent on correction of chloride transport abnormalities. However, it is now clear that the pathophysiology of CF airways disease is far more complex than can be solely attributed to altered chloride permeability. For example, in addition to functioning as a chloride channel, CFTR also has been implicated in the regulation of other apical membrane conductance pathways through interactions with the amiloride sensitive epithelial sodium channel (ENaC) and the outwardly rectifying chloride channel (ORCC). Superimposed on this functional diversity of CFTR is a highly regulated pattern of CFTR expression in the lung. This heterogeneity occurs at both the level of CFTR protein expression within different cell types in the airway and the anatomical location of these cells in the lung. Potential targets for gene therapy of CF include ciliated, non-ciliated, and goblet cells in the surface airway epithelium as well as submucosal glands within the interstitium of the airways. Each of these distinct cellular compartments may have functionally distinct roles in processes which affect the pathogenesis of CF airways disease, such as fluid and electrolyte balance. However, it is presently unclear which of these cellular targets are most pathophysiologic relevant with regard to gene therapy. Elucidation of the underlying mechanisms of CFTR function in the airway will allow for the rational design of gene therapy approaches for CF lung diseases. This review will provide a summary of the field's current knowledge regarding CFTR functional diversity in the airway and the implications of such diversity for gene therapies of CF lung disease.     

Mutation analysis and loss of heterozygosity of PEDF in central nervous system primitive neuroectodermal tumors

CF mice, i.e., mice without functional CFTR (cystic fibrosis transmembrane conductance regulator) exhibit a very low basal Isc in all regions of the intestinal tract. The low basal Isc in the intestinal epithelia of the CF mice appears to be a result of lack of spontaneous Cl- secretion (and possibly HCO3- secretion) mediated by neurotransmitter release from the enteric nervous system. In contrast to intestinal epithelia from normal mice, the intestinal epithelia of CF mice do not secrete Cl- in response to agents that increase cAMP (forskolin). Furthermore, as in human CF patients, agents that increase intracellular Ca2+ (bethanacol, ionomycin) failed to elicit Cl- secretion in the intestinal epithelia of CF mice. There was no difference in the electrogenic Na(+)-coupled glucose absorption in the CF murine jejuna compared to jejuna from normal mice. However, further studies are warranted to determine whether amiloride-sensitive Na+ absorption is upregulated in the murine CF colon. It was concluded that the intestinal epithelium of the CF mouse model exhibits some striking similarities to its human counterpart, and therefore should be very useful in further characterizing the ion transport defects in this disease.     

Effect of deletion mutations on the function of CFTR chloride channels

Mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a unique R domain; CFTR functions as a Cl- channel regulated by phosphorylation and by nucleoside triphosphates. To study the domains of CFTR involved in Cl- channel function, we expressed mutants lacking various domains and assayed cAMP-stimulated Cl- channel activity using the halide-sensitive fluorophore, 6-methoxy-N-(3'-sulfopropyl)-quinolinium. We previously reported that deletion of part of the R domain (residues 708-835) produced Cl- channels that were constitutively open. Here we show that more extensive deletions within the R domain failed to generate functional CFTR Cl- channels; the portion of protein that could be deleted without destroying function corresponds to sequences that are not conserved in related proteins. In contrast, when we deleted the two NBDs (either alone, together, or in combination with the R domain), we did not observe functional Cl- channels. CFTR has a unique carboxyl terminus that is conserved across species. However, truncation of the carboxyl terminus (up to, but not including, NBD2) produced a regulated anion permeability similar to that of wild-type CFTR, suggesting that this region is not essential for channel function. Expression of two CF-associated nonsense mutants (G542X and W1316X) also failed to generate functional CFTR Cl- channels. These results help define structure:function relationships for CFTR and identify the domains that are required for Cl- channel function.     

A micromechanical model of lung tissue rheology

We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl- secretion (Isc) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent Isc in a concentration-dependent manner, having an apparent Ki of 142 microM. Salicylic acid inhibited Isc with an apparent Ki of 646 microM. We determined whether ibuprofen would also inhibit the forskolin-stimulated Isc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 microM) inhibited the forskolin-induced Isc in MTEs and HBEs by 59+/-4% (n = 11) and 39+/-6% (n = 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl- (ICl) and basolateral K+ (IK) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 microM) reduced both the ICl and IK; reducing ICl and IK by 60 and 15%, respectively. To determine whether this inhibition of ICl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl- channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 microM) reduced CFTR Cl- current by 60+/-16% and this was explained by a short-lived block (approximately 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07+/-0.04 pA to 0.59+/-0.04 pA (n = 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl- current by 50+/-8% with an apparent reduction in single channel amplitude from 1.08+/-0.03 pA to 0.48+/-0.06 pA (n = 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl- secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl- channels as well as basolateral membrane K+ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia.     

An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton

The function of the cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl- channel in the apical membrane of epithelial cells is extensively documented. However, less is known about the molecular determinants of CFTR residence in the apical membrane, basal regulation of its Cl- channel activity, and its reported effects on the function of other transporters. These aspects of CFTR function likely require specific interactions between CFTR and unknown proteins in the apical compartment of epithelial cells. Here we report that CFTR interacts with the recently discovered protein, EBP50 (ERM-binding phosphoprotein 50). EBP50 is concentrated at the apical membrane in human airway epithelial cells, in vivo, and CFTR and EBP50 associate in in vitro binding assays. The CFTR-EBP50 interaction requires the COOH-terminal DTRL sequence of CFTR and utilizes either PDZ1 or PDZ2 of EBP50, although binding to PDZ1 is of greater affinity. Through formation of a complex, the interaction between CFTR and EBP50 may influence the stability and/or regulation of CFTR Cl- channel function in the cell membrane and provides a potential mechanism through which CFTR can affect the activity of other apical membrane proteins.     

An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer

Cystic fibrosis (CF) is an inherited disease of epithelial cell ion transport that is associated with pathology in multiple organ systems, including lung, pancreas, and liver. As treatment of the pulmonary manifestations of CF has improved, management of CF liver disease has become increasingly important in adult patients. This report describes an approach for treating CF liver disease by somatic gene transfer. In situ hybridization and immunocytochemistry analysis of rat liver sections indicated that the endogenous CFTR (cystic fibrosis transmembrane conductance regulator) gene is primarily expressed in the intrahepatic biliary epithelial cells. To specifically target recombinant genes to the biliary epithelium in vivo, recombinant adenoviruses expressing lacZ or human CFTR were infused retrograde into the biliary tract through the common bile duct. Conditions were established for achieving recombinant gene expression in virtually all cells of the intrahepatic bile ducts in vivo. Expression persisted in the smaller bile ducts for the duration of the experiment, which was 21 days. These studies suggest that it may be feasible to prevent CF liver disease by genetically reconstituting CFTR expression in the biliary tract, using an approach that is clinically feasible.     

Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) contains multiple membrane spanning sequences that form a Cl- channel pore and cytosolic domains that control the opening and closing of the channel. The fourth intracellular loop (ICL4), which connects the tenth and eleventh transmembrane spans, has a primary sequence that is highly conserved across species, is the site of a preserved sequence motif in the ABC transporter family, and contains a relatively large number of missense mutations associated with cystic fibrosis (CF). To investigate the role of ICL4 in CFTR function and to learn how CF mutations in this region disrupt function, we studied several CF-associated ICL4 mutants. We found that most ICL4 mutants disrupted the biosynthetic processing of CFTR, although not as severely as the most common DeltaF508 mutation. The mutations had no discernible effect on the channel's pore properties; but some altered gating behavior, the response to increasing concentrations of ATP, and stimulation in response to pyrophosphate. These effects on activity were similar to those observed with mutations in the nucleotide-binding domains, suggesting that ICL4 might help couple activity of the nucleotide-binding domains to gating of the Cl- channel pore. The data also explain how these mutations cause a loss of CFTR function and suggest that some patients with mutations in ICL4 may have a milder clinical phenotype because they retain partial activity of CFTR at the cell membrane.     

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.     

Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A

Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl- channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 microM) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of >15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is approximately 60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.     

Infection with hepatitis G virus and its strain variant, the GB agent (GBV-C), among blood donors in Japan

Much of the morbidity and mortality seen in cystic fibrosis (CF) is related to chronic infection of the respiratory tract with Pseudomonas aeruginosa. Some studies have attributed the strong relationship between CF and Pseudomonas colonization to the presence of increased numbers of specific cell-surface receptors, although other work suggests that this relates to the presence of mucus. Several groups are now assessing the use of gene transfer as a novel form of treatment for CF. We have examined whether P. aeruginosa binding to freshly obtained CF respiratory epithelial cells is increased, and have studied the effects of transfer of the CF transmembrane conductance regulator (CFTR) gene on this attachment. Binding of P. aeruginosa to noncultured nasal epithelial cells from both CF patients (n = 31) and healthy controls (n = 15) was studied with scanning electron microscopy. Binding was also assessed for CF cells following transfection with CFTR/liposome complexes. Epifluorescence microscopy was used to assess the effects of gene transfer on chloride fluxes. Adherence of P. aeruginosa directly to the cell surface of CF airway epithelium was significantly (P < 0.001) increased over that in non-CF controls. Liposome-mediated CFTR gene transfer resulted in a significant (P < 0.01) reduction in the numbers of bacteria bound to ciliated epithelial cells. Fluorescence microscopy confirmed correction of the basic chloride defect. Thus, in CF, the absence of normal CFTR results in increased binding of P. aeruginosa to respiratory epithelial cells. This abnormality can be corrected in vitro by restoration of CFTR function. This has important implications both for the pathogenesis of CF and for the future application and assessment of gene therapy for this disease.