miR-16 rescues F508del-CFTR function in native cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is due to mutations in the CFTR gene, which prevents correct folding, trafficking and function of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. The dysfunctional effect of CFTR mutations, principally the F508del-CFTR mutant, is further manifested by hypersecretion of the pro-inflammatory chemokine interleukin-8 into the airway lumen, which further contributes to morbidity and mortality. We have hypothesized that microRNA (miR)-based therapeutics could rescue the dysfunctional consequences of mutant CFTR. Here we report that a miR-16 mimic can effectively rescue F508del-CFTR protein function in airway cell lines and primary cultures, of differentiated human bronchial epithelia from F508del homozygotes, which express mutant CFTR endogenously. We also identify two other miRs, miR-1 and miR-302a, which are also active. Although miR-16 is expressed at basal comparable levels in CF and control cells, miR-1 and miR-302a are undetectable. When miR mimics are expressed in CF lung or pancreatic cells, the expression of the F508del-CFTR protein is significantly increased. Importantly, miR-16 promotes functional rescue of the cyclic AMP-activated apical F508del-CFTR chloride channel in primary lung epithelial cells from CF patients. We interpret these findings to suggest that these miRs may constitute novel targets for CF therapy.

Mediastinal tuberculosis in Bradford, United Kingdom: the role of mediastinoscopy.

OBJECTIVE: To evaluate patients with mediastinal tuberculosis (MT), their demographic profiles, symptoms, radiological features and the role of mediastinoscopy. METHODS: This retrospective study conducted at Bradford Teaching Hospitals, Bradford, United Kingdom, looked at the case notes of 160 (13%) patients with MT out of a cohort of 1252 notifications of tuberculosis (TB) cases from 1995 to 2004, analysing the demographic data, diagnostic findings, computed tomography (CT) scans and outcomes. Interventions included bronchoscopy, lymph node biopsy and mediastinoscopy. RESULTS: Patient age ranged from 1 to 75 years; the majority were females and from minority ethnic groups. Contact history was positive in 76% of cases. Cough was the most common symptom (50%); however, asymptomatic patients were also common (45%). Heaf test was positive in 99%. Right paratracheal lymphadenopathy was common on chest X-ray and chest CT scan. Mediastinoscopy was performed in only 37 patients with definitive diagnosis. CONCLUSION: MT should be suspected in adult asymptomatic immigrants presenting with mediastinal adenopathy and a strongly positive Heaf test. Trial of anti-tuberculosis treatment should be initiated and response should guide further management. Mediastinoscopy is required in only a minority of patients.

Absorptive clearance of DTPA as an aerosol-based biomarker in the cystic fibrosis airway.

Biomarkers providing in vivo quantification of the basic elements of cystic fibrosis (CF) lung disease are needed. A study was performed to determine whether the absorption of a small radiolabelled hydrophilic molecule (Indium-111 (In-)DTPA) would be increased in CF airways. DTPA clearance has been used previously to assess epithelial permeability and may also be useful for quantifying liquid absorption. The absorptive clearance rate of DTPA was quantified in 10 CF and 11 control subjects using a novel aerosol technique. Subjects inhaled an aerosol containing nonabsorbable technetium-99m sulfur colloid (Tc-SC) particles and In-DTPA. Tc-SC clearance from the lung is exclusively mucociliary, while In-DTPA is cleared by both absorption and mucociliary clearance. The difference between the In-DTPA and Tc-SC clearance rates estimates In-DTPA absorption. Tc-SC (mucociliary) clearance was similar in central and peripheral zones in CF and non-CF lungs. Total In-DTPA clearance was increased in both zones in CF lungs. The absorptive component of In-DTPA clearance was increased in the airway-dominated central lung zones in CF (42% x h( -1) versus 32% x h(-1), p = 0.03). The absorption of In-DTPA is increased in the CF airway. Further study is needed to understand the relative roles of fluid absorption, inflammation and other mechanisms potentially affecting epithelial permeability and DTPA absorption.

Electrogenic bicarbonate secretion by prairie dog gallbladder.

Pathological rates of gallbladder salt and water transport may promote the formation of cholesterol gallstones. Because prairie dogs are widely used as a model of this event, we characterized gallbladder ion transport in animals fed control chow by using electrophysiology, ion substitution, pharmacology, isotopic fluxes, impedance analysis, and molecular biology. In contrast to the electroneutral properties of rabbit and Necturus gallbladders, prairie dog gallbladders generated significant short-circuit current (I(sc); 171 +/- 21 microA/cm(2)) and lumen-negative potential difference (-10.1 +/- 1.2 mV) under basal conditions. Unidirectional radioisotopic fluxes demonstrated electroneutral NaCl absorption, whereas the residual net ion flux corresponded to I(sc). In response to 2 microM forskolin, I(sc) exceeded 270 microA/cm(2), and impedance estimates of the apical membrane resistance decreased from 200 Omega.cm(2) to 13 Omega.cm(2). The forskolin-induced I(sc) was dependent on extracellular HCO(3)(-) and was blocked by serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS) and acetazolamide, whereas serosal bumetanide and Cl(-) ion substitution had little effect. Serosal trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman and Ba(2+) reduced I(sc), consistent with the inhibition of cAMP-dependent K(+) channels. Immunoprecipitation and confocal microscopy localized cystic fibrosis transmembrane conductance regulator protein (CFTR) to the apical membrane and subapical vesicles. Consistent with serosal DNDS sensitivity, pancreatic sodium-bicarbonate cotransporter protein pNBC1 expression was localized to the basolateral membrane. We conclude that prairie dog gallbladders secrete bicarbonate through cAMP-dependent apical CFTR anion channels. Basolateral HCO(3)(-) entry is mediated by DNDS-sensitive pNBC1, and the driving force for apical anion secretion is provided by K(+) channel activation.

Methods for detecting internalized, FM 1-43 stained particles in epithelial cells and monolayers.

The membrane dye FM 1-43 has frequently been used to quantify exocytosis in neurons. In epithelia, intense lateral intracellular space staining and fluctuations in baseline labeling produced inconsistent results. Membrane retrieved in the presence of FM 1-43 retains the dye, however, and cells that undergo compensatory endocytosis during and following evoked exocytosis contain punctate, fluorescent particles after washout of external stain. As an alternative measure of trafficking, we quantified the fluorescent puncta retained after dye washout and tested our method on both coverslip-grown cell clusters and filter-grown intact monolayers. Images for analysis were acquired using serial sectioning with either epifluorescence or confocal microscopy. Tests with an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that 1), the algorithm identified the same number of internalized particles with either epifluorescence or confocal microscopy acquired images; 2), low density clusters exhibited significantly more internalized particles per cell than either filter-grown monolayers or high density clusters; 3), ATP stimulation significantly increased the number of internalized particles in all preparations; and 4), the number of particles internalized was comparable to capacitance measurements of exocytosis. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells.

PKA-dependent ENaC trafficking requires the SNARE-binding protein complexin.

Acute regulation of epithelial sodium channel (ENaC) function at the apical surface of polarized kidney cortical collecting duct (CCD) epithelial cells occurs in large part by changes in channel number, mediated by membrane vesicle trafficking. Several soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) have been implicated in this process. A novel SNARE-binding protein, complexin, has been identified in nervous tissue which specifically binds to and stabilizes SNARE complexes at synaptic membranes to promote vesicle fusion. To test whether this protein is present in mouse CCD (mCCD) cells and its possible involvement in acute ENaC regulation, we cloned complexin (isoform II) from a mouse kidney cDNA library. Complexin II mRNA coexpressed with alpha-, beta-, and gamma-ENaC subunits in Xenopus laevis oocytes reduced sodium currents to 16 +/- 3% (n = 19) of control values. Short-circuit current (I(sc)) measurements on mCCD cell lines stably over- or underexpressing complexin produced similar results. Basal I(sc) was reduced from 12.0 +/- 1.0 (n = 15) to 2.0 +/- 0.4 (n = 15) and 1.8 +/- 0.3 (n = 17) microA/cm(2), respectively. Similarly forskolin-stimulated I(sc) was reduced from control values of 20.0 +/- 2 to 2.7 +/- 0.5 and 2.3 +/- 0.4 microA/cm(2) by either increasing or decreasing complexin expression. Surface biotinylation demonstrated that the complexin-induced reduction in basal I(sc)was due to a reduction in apical membrane-resident ENaC and the inhibition in forskolin stimulation was due to the lack of ENaC insertion into the apical membrane to increase surface channel number. Immunofluorescent localization of SNARE proteins in polarized mCCD epithelia detected the presence of syntaxins 1 and 3 and synaptosomal-associated protein of 23 kDa (SNAP-23) at the apical membrane, and vesicle-associated membrane protein (VAMP2) was localized to intracellular compartments. These findings identify SNAREs that may mediate ENaC-containing vesicle insertion in mCCD epithelia and suggest that stabilization of SNARE interactions by complexin is an essential aspect of the regulated trafficking events that increase apical membrane ENaC density either by constitutive or regulated trafficking pathways.

Established cell lines used in cystic fibrosis research.

The development of immortalized cell lines has been a significant benefit to the study of human disease, due to limitations in using primary cells and the availability of tissue. The immortalization of cells from cystic fibrosis (CF) patients as well as cells from non-CF individuals from tissues relevant to CF has been critical to enhancing our understanding of the physiological, biochemical and genetic mechanisms underlying CF and for the development of therapeutic strategies designed to manage CF pathology. A comprehensive list of immortalized cells from various tissue and species, with an emphasis on epithelial cells, is presented and discussed here.

Predominant VNTR family of strains of Mycobacterium tuberculosis isolated from South Asian patients.

SETTING: Despite the low incidence of tuberculosis in the UK, some minority ethnic groups, particularly those originating from South Asia, experience very high incidence rates. OBJECTIVE: Comparison of the variable number tandem repeat (VNTR) profiles of strains of Mycobacterium tuberculosis circulating in an immigrant community in the UK with those found in the country of ethnic origin. DESIGN: Isolates of M. tuberculosis were collected from samples obtained from patients attending clinics in Leeds and Bradford, UK and Rawalpindi, Pakistan. Strains were compared using VNTR analysis and mixed-linker PCR. RESULTS: Comparison of VNTR profiles found that one profile (42235) represented 37% of patient isolates from Rawalpindi and 23% of patient isolates in Leeds and Bradford, where it was associated exclusively with patients with South Asian names. A second profile (02235) represented 15% of patient isolates in Leeds and Bradford, and was also exclusively associated with the South Asian community. These profiles could be subdivided by mixed-linker PCR analysis. CONCLUSION: The VNTR profile 42235 may represent a family of strains commonly found in communities associated with South Asia.

Microelectrode and impedance analysis of anion secretion in Calu-3 cells.

Calu-3 cells secrete HCO(3)(-) in response to cAMP agonists but can be stimulated to secrete Cl(-) with K(+) channel activating agonists. Microelectrode and impedance analysis experiments were performed to obtain a better understanding of the conductances and driving forces involved in these different modes of anion secretion in Calu-3 cells. Microelectrode studies revealed apical and basolateral membrane depolarizations upon the addition of forskolin (V(ap) -52 mV vs. -21 mV; V(bl) -60 mV vs. -44 mV) that paralleled the hyperpolarization of the mucosal negative transepithelial voltage (V(T) -8 mV vs. -23 mV). These changes were accompanied by a decrease in the apical membrane fractional resistance (F(Rap)) from approximately 0.50 to 0.08, consistent with the activation of an apical membrane conductance. The subsequent addition of 1-ethyl-2-benzimidazolinone (1-EBIO), a K(+) channel activator, hyperpolarized V(ap) to -27 mV, V(bl) to -60 mV and V(T) to -33 mV. Impedance analysis revealed the apical membrane resistance (R(ap)) of the forskolin-stimulated cells was less than 20 ohm cm(2), indeed in most monolayers R(ap) fell to less than 5 ohm cm(2). The impedance derived estimate of the basolateral membrane resistance (R(bl)) was approximately 170 ohm cm(2) in forskolin treated cells and fell to 50 ohm cm(2) with the addition of 1-EBIO. Using these values for the R(bl) and the F(Rap) value of 0.08 yields a R(ap) of approximately 14 ohm cm(2) in the presence of forskolin and 4 ohm cm(2) in the presence of forskolin plus 1-EBIO. Thus, by two independent methods, forskolin-stimulated Calu-3 cells are seen to have a very high apical membrane conductance of 50 to 200 mS/cm(2). Therefore, we would assert that even at one-tenth the anion selectivity for Cl(-), this high conductance could support the conductive exit of HCO(3)(-) across the apical membrane. We further propose that this high apical membrane conductance serves to clamp the apical membrane potential near the equilibrium potential for Cl(-) and thereby provides the driving force for HCO(3)(-) secretion in forskolin-stimulated Calu-3 cells. The hyperpolarization of V(ap) and V(bl) caused by 1-EBIO provides a driving force for Cl(-) exit across the apical membrane, inhibits the influx of HCO(3)(-) on the Na(+):HCO(3)(-) cotransporter across the basolateral membrane, activates the basolateral membrane Na(+):K:2Cl(-) cotransporter and thereby provides the switch from HCO(3)(-) secretion to Cl(-) secretion.

Role of snare proteins in CFTR and ENaC trafficking.

The apical membrane ion channels, CFTR and ENaC, undergo regulated trafficking as a means of controlling their plasma membrane density. This provides a mechanism for regulating the Cl and Na conductance properties of epithelial apical membranes, and thus the transepithelial ion transport rates. Physical and functional interactions between these channels and SNARE proteins, in particular syntaxin 1A (S1A), provide a mechanism for linking the known vesicle fusion machinery with this process. In this paper we summarize evidence indicating that the interaction of S1A with CFTR and ENaC reduces channel currents in a syntaxin-isoform-specific manner. The acute cAMP-regulated CFTR trafficking event, which is reported by an increase in membrane capacitance in response to cAMP, is also inhibited by exogenous S1A expression. We tagged both channels with flag epitopes on their extracellular surfaces to monitor their plasma membrane expression as a function of S1A co-expression. The data indicate that the reduction in current caused by S1A is associated with a marked decrease in the amount of CFTR or ENaC detected at the cell surface. These findings suggest that S1A inhibits ion channel insertion into the plasma membrane, either by disrupting the stoichiometry of SNARE protein associations that mediate channel trafficking, or by physically associating with the channels to prevent their insertion. These data link the SNARE machinery to the regulation of apical membrane ion channel density, and suggest that phosphorylation-dependent interactions of these channels with SNARE proteins may acutely regulate this process.

E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells.

Although it is generally recognized that cystic fibrosis transmembrane conductance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at its COOH terminus, the identity of the PDZ domain protein(s) that interact with CFTR is uncertain, and the functional impact of this interaction is not fully understood. By using human airway epithelial cells, we show that CFTR associates with Na(+)/H(+) exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/NHE regulatory factor (NHERF)-related PDZ domain protein. The PDZ binding motif located at the COOH terminus of CFTR interacts preferentially with the second PDZ domain of E3KARP, with nanomolar affinity. In contrast to EBP50/NHERF, E3KARP is predominantly localized (>95%) in the membrane fractions of Calu-3 and T84 cells, where CFTR is located. Moreover, confocal immunofluorescence microscopy of polarized Calu-3 monolayers shows that E3KARP and CFTR are co-localized at the apical membrane domain. We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl(-) currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actin-binding protein, the formation of a CFTR.E3KARP.ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells.

Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells.

Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a subapical pool of CFTR into the plasma membrane (PM). However, this issue has been controversial, in part because of the difficulty in distinguishing cell surface vs. intracellular CFTR. Recently, a fully functional, epitope-tagged form of CFTR (M2-901/CFTR) that can be detected immunologically in nonpermeabilized cells was characterized (Howard M, Duvall MD, Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J Physiol Cell Physiol 269: C1565-C1576, 1995; and Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. Am J Physiol Cell Physiol 273: C2080-C2089, 1997). We have developed replication-defective recombinant adenoviruses that express M2-901/CFTR and used them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells. Virally expressed M2-901/CFTR was functional and was readily detected on the apical surface of FSK-stimulated polarized MDCK cells. Interestingly, at low multiplicity of infection, we observed FSK-stimulated insertion of M2901/CFTR into the apical PM, whereas at higher M2-901/CFTR expression levels, no increase in surface expression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2-901/CFTR redistribution to the PM upon FSK stimulation and demonstrates that the apically inserted M2-901/CFTR originates from a population of subapical vesicles. Our observations may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.

Mechanisms underlying regulated CFTR trafficking.

Stimulation of membrane capacitance and cell surface labeling of epitope-tagged CFTR provide evidence of cAMP-regulated CFTR trafficking. Co-expression of syntaxin 1A inhibits cAMP-stimulated current and capacitance changes in CFTR expressing cells and blocks cAMP-induced increases in cell surface CFTR. Inhibition of CFTR trafficking by syntaxin over-expression suggests a role for SNARE proteins in this process. CFTR phosphorylation may alter physical interactions with SNARE proteins to regulate plasma membrane CFTR density.

Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.

Regulation of the amiloride-sensitive epithelial sodium channel by syntaxin 1A.

The first step in transepithelial sodium absorption lies at the apical membrane where the amiloride-sensitive, epithelial sodium channel, ENaC, facilitates sodium entry into the cell. Here we report that the vesicle traffic regulatory (SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)) protein, syntaxin 1A (S1A), inhibits ENaC mediated sodium entry. This inhibitory effect is selective for S1A and is not reproduced by syntaxin 3. The inhibition does not require the membrane anchoring domain of syntaxin 1A. It was reversed by the S1A-binding protein, Munc-18, but not by a Munc-18 mutant, which lacks syntaxin affinity. Immunostaining of epitope-tagged ENaC subunits showed that syntaxin 1A decreases ENaC current by reducing the number of ENaC channels in the plasma membrane; S1A does not interfere with ENaC protein expression. Immunoprecipitation of syntaxin 1A from the sodium-transporting epithelial cell line, A6, co-precipitates ENaC. These findings indicate that syntaxin 1A and other members of the SNARE machinery are involved in the control of plasma membrane ENaC content, and they suggest that SNARE proteins participate in the regulation of sodium absorption in relation to agonist mediated vesicle insertion-retrieval processes.

Syntaxin 1A inhibits regulated CFTR trafficking in xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial cell Cl channel, whose gating activity and membrane trafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett, D. J. Benos, M. W. Quick, and K. L. Kirk. Nature 390: 302-305, 1997), a protein best known for its role in membrane trafficking and neurosecretion. To examine the mechanism of syntaxin 1A inhibition, we expressed these proteins in Xenopus oocytes and monitored agonist-induced changes in plasma membrane capacitance and cell surface fluorescence of CFTR that contains an external epitope tag. cAMP stimulation elicited large increases in membrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTR content. Injection of botulinum toxin/C1 rapidly reversed syntaxin's effects on current and capacitance, indicating that they cannot be explained by an effect on CFTR synthesis. Functional expression of other integral membrane proteins, including Na-coupled glucose transporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotide receptor, and viral hemagglutinin protein, was not affected by syntaxin 1A coexpression. These findings indicate that acute regulation of the number of CFTR Cl channels in plasma membrane is one mechanism by which cAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTR content by syntaxin 1A is consistent with the concept that syntaxin and other components of the SNARE machinery are involved in regulated trafficking of CFTR.

Rescue of dysfunctional deltaF508-CFTR chloride channel activity by IBMX.

Nucleotide-dependent gating of DeltaF508-CFTR was evaluated in membrane patches excised from HEK 293 and mouse L-cells and compared to observations on wt-CFTR channels recorded in the same expression systems. DeltaF508-CFTR exhibited PKA activated, ATP-dependent channel gating. When compared to wt-CFTR, the Km for ATP was increased by ninefold (260 micron vs. 28 micron) and maximal open probability (Po) was reduced by 49% (0.21 +/- 0.06 vs. 0.41 +/- 0. 02). Additionally, in the absence of PKA, DeltaF508-CFTR inactivated over a 1 to 5 min period whereas wt-CFTR remained active. Time-dependent inactivation could be mimicked in wt-CFTR by the intermittent absence of ATP in the cytosolic solution. The effects of 3-isobutyl-1-methyl xanthine (IBMX), a compound reported to stimulate DeltaF508-CFTR, were evaluated on wt- and DeltaF508-CFTR channels. At concentrations up to 5 mm, IBMX caused a concentration dependent reduction in the observed single channel amplitude (i) of wt-CFTR (maximal observed reduction 35 +/- 3%). However, IBMX failed to significantly alter total patch current because of a concomitant 30% increase in Po. The effects of IBMX on DeltaF508-CFTR were similar to effects on wt-CFTR in that i was reduced and Po was increased by similar magnitudes. Additionally, DeltaF508-CFTR channel inactivation was dramatically slowed by IBMX. These results suggest that IBMX interacts with the ATP-bound open state of CFTR to introduce a short-lived nonconducting state which prolongs burst duration and reduces apparent single channel amplitude. A secondary effect observed in DeltaF508-CFTR, which may result from this interaction, is a prolongation of the activated state. In light of previously proposed linear kinetic models of CFTR gating, these results suggest that IBMX traps CFTR in an ATP-bound state which may preclude inactivation of DeltaF508-CFTR.

Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.

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.