Pathogenesis of cystic fibrosis airways disease.
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Bacterial Infections↗
Biomedical subjects
R C Boucher
Publications and source records attributed to R C Boucher.
Regulation and functional significance of airway surface liquid pH.
In gastrointestinal tissues, cumulative evidence from both in vivo and in vitro studies suggests a role for the cystic fibrosis transmembrane conductance regulator (CFTR) in apical epithelial bicarbonate conductance. Abnormal lumenal acidification is thus hypothesized to play a role in the genesis of cystic fibrosis (CF) pancreatic disease. However, consensus regarding CFTR's participation in pH regulation of airway surface liquid (ASL) and thus the contribution of ASL pH to the etiology of CF lung disease, is lacking. The absence of data reflects difficulties in both sampling ASL in vivo and modeling ASL biology in vitro. Here we evaluate the evidence in support of a lumenal acidification hypothesis in the CF lung, summarize current knowledge of pH regulation in the normal airway and illustrate how hyper-acidified airway secretions could contribute to the pathogenesis of CF lung disease.
Animals↗
Permeabilization via the P2X7 purinoreceptor reveals the presence of a Ca2+-activated Cl- conductance in the apical membrane of murine tracheal epithelial cells.
Calcium-activated Cl(-) secretion is an important modulator of regulated ion transport in murine airway epithelium and is mediated by an unidentified Ca(2+)-stimulated Cl(-) channel. We have transfected immortalized murine tracheal epithelial cells with the cDNA encoding the permeabilizing P2X(7) purinoreceptor (P2X(7)-R) to selectively permeabilize the basolateral membrane and thereby isolate the apical membrane Ca(2+)-activated Cl(-) current. In P2X(7)-R-permeabilized cells, we have demonstrated that UTP stimulates a Cl(-) current across the apical membrane of CF and normal murine tracheal epithelial cells. The magnitude of the UTP-stimulated current was significantly greater in CF than in normal cells. Ion substitution studies demonstrated that the current exhibited a permselectivity sequence of Cl(-) > I(-) > Br(-) > gluconate(-). We have also determined a rank order of potency for putative Cl(-) channel blockers: niflumic acid > or = 5-nitro-2-(3-phenylpropylamino)benzoic acid > 4, 4'-diisothiocyanostilbene-2,2'-disulfonate > glybenclamide >> diphenlyamine-2-carboxylate, tamoxifen, and p-tetra-sulfonato-tetra-methoxy-calix[4]arene. Complete characterization of this current and the corresponding single channel properties could lead to the development of a new therapy to correct the defective airway surface liquid in cystic fibrosis patients.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations.
Nucleotides are important extracellular signaling molecules. At least five mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP, or UDP. Although the existence of ectoenzymes that metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. Therefore, we have studied the kinetics of accumulation and metabolism of endogenous ATP in the extracellular medium of four different cell lines. ATP concentrations reached a maximum immediately after change of medium and decreased thereafter with a single exponential decay (t(1/2);1 approximately;230-40 min). ATP levels did not fall to zero but attained a base-line concentration that was independent of the medium volume and of the initial ATP concentration. Although the base-line concentration of ATP remained stable for up to 12 h, [gamma-(32)P]ATP added to resting cells as a radiotracer was completely degraded within 120 min, indicating that steady state reflected a basal rate of ATP release balanced by ATP hydrolysis (20-200 fmol x min(-)(1) x cell(-)(6)). High performance liquid chromatography analysis revealed that the gamma-phosphate of ATP was rapidly, although transiently, transferred during steady state to species subsequently identified as UTP and GTP, indicating the existence of both ecto-nucleoside diphosphokinase activity and the accumulation of endogenous UDP and GDP. Conversely, addition of [gamma-(32)P]UTP to resting cells resulted in transient formation of [gamma-(32)P]ATP, indicating phosphorylation of endogenous ADP by nucleoside diphosphokinase. The final (32)P-products of [gamma-(32)P]ATP metabolism were [(32)P]orthophosphoric acid and a (32)P-labeled species that was further purified and identified as [(32)P]inorganic pyrophosphate. In C6 cells, the formation of [(32)P]pyrophosphate from [gamma-(32)P]ATP at steady state exceeded by 3-fold that of [(32)P]orthophosphate. These results illustrate for the first time a constitutive release of ATP and other nucleotides and reveal the existence of a complex extracellular metabolic pathway for released nucleotides. In addition to the existence of an ecto-ATPase activity, our results suggest a major scavenger role of ecto-ATP pyrophosphatase and a transphosphorylating activity of nucleoside diphosphokinase.
Adenosine Triphosphate↗
Cell to cell communication in response to mechanical stress via bilateral release of ATP and UTP in polarized epithelia.
Airway epithelia are positioned at the interface between the body and the environment, and generate complex signaling responses to inhaled toxins and other stresses. Luminal mechanical stimulation of airway epithelial cells produces a propagating wave of elevated intracellular Ca(2+) that coordinates components of the integrated epithelial stress response. In polarized airway epithelia, this response has been attributed to IP(3) permeation through gap junctions. Using a combination of approaches, including enzymes that destroy extracellular nucleotides, purinergic receptor desensitization, and airway cells deficient in purinoceptors, we demonstrated that Ca(2+) waves induced by luminal mechanical stimulation in polarized airway epithelia were initiated by the release of the 5' nucleotides, ATP and UTP, across both apical and basolateral membranes. The nucleotides released into the extracellular compartment interacted with purinoceptors at both membranes to trigger Ca(2+) mobilization. Physiologically, apical membrane nucleotide-release coordinates airway mucociliary clearance responses (mucin and salt, water secretion, increased ciliary beat frequency), whereas basolateral release constitutes a paracrine mechanism by which mechanical stresses signal adjacent cells not only within the epithelium, but other cell types (nerves, inflammatory cells) in the submucosa. Nucleotide-release ipsilateral and contralateral to the surface stimulated constitutes a unique mechanism by which epithelia coordinate local and distant airway defense responses to mechanical stimuli.
Adenosine Triphosphate↗
Human gene marker/therapy clinical protocols.
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Clinical Protocols↗
Safety and biological efficacy of a lipid-CFTR complex for gene transfer in the nasal epithelium of adult patients with cystic fibrosis.
Gene transfer is an attractive option to treat the basic defect in cystic fibrosis. In a double-blind, placebo-controlled, rising-dose tolerance study in the nasal epithelium, we tested the safety and efficacy of a cationic liposome [p-ethyl-dimyristoylphosphadityl choline (EDMPC) cholesterol] complexed with an expression plasmid containing hCFTR cDNA. Eleven adult CF patients were studied in a protocol that allowed comparisons within individual subjects: vector and placebo were sprayed into alternate nostrils at intervals over 7 h. After dosing, vector-specific DNA was present in nasal lavage of all subjects for up to 10 days. There were no adverse events. The vector-treated epithelium did not exhibit a significant increase in CFTR-mediated Cl- conductance from baseline and was not different from the placebo-treated nostril: mean deltaCFTR Cl- conductance, mV +/- SEM, -1.6+/-0.4 vs -0.6+/-0.4, respectively. CFTR-mediated Cl- conductance increased toward normal during repetitive nasal potential difference measurements over the 3 days before dosing which influenced the postdosing calculations. No vector-specific mRNA was detected in the nasal epithelial scrape biopsies, although endogenous CFTR mRNA was detected in all subjects. We conclude that the lipid-DNA complex is safe, but did not produce consistent evidence of gene transfer to the nasal epithelium by physiologic or molecular measures.
Adult↗
G-protein-coupled receptors as targets for gene transfer vectors using natural small-molecule ligands.
Gene therapy for cystic fibrosis (CF) has focused on correcting electrolyte transport in airway epithelia. However, success has been limited by the failure of vectors to attach and enter into airway epithelia, and may require redirecting vectors to targets on the apical membrane of airway cells that mediate these functions. The G-protein-coupled P2Y2 receptor (P2Y2-R) is abundantly expressed on the airway lumenal surface and internalizes into coated pits upon agonist activation. We tested whether a small-molecule-agonist (UTP) could direct vectors to P2Y2-R and mediate attachment, internalization, and gene transfer. Fluorescein-UTP studies demonstrated that P2Y2-R agonists internalized with their receptor, and biotinylated UTP (BUTP) mediated P2Y2-R-specific internalization of fluorescently labeled streptavidin (SAF) or SAF conjugated to biotinylated Cy3 adenoviral-vector (BCAV). BUTP conjugated to BCAV mediated P2Y2-R-specific gene transfer in (1) adenoviral-resistant A9 and polarized MDCK cells by means of heterologous P2Y2-R, and (2) well-differentiated human airway epithelial cells by means of endogenous P2Y2-R. Targeting vectors with small-molecule-ligands to apical membrane G-protein-coupled receptors may be a feasible approach for successful CF gene therapy.
Adenoviridae↗
Pseudotyped human lentiviral vector-mediated gene transfer to airway epithelia in vivo.
We used a replication defective human lentiviral (HIV) vector encoding the lacZ cDNA and pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein (G) to evaluate the utility of this vector system in airway epithelia. In initial studies, apical application of vector to polarized well differentiated human airway epithelial cell cultures produced minimal levels of transgene expression whereas basolateral application of vector enhanced levels of transduction approximately 30-fold. Direct in vivo delivery of HIV vectors to the nasal epithelium and tracheas of mice failed to mediate gene transfer, but injury with sulfur dioxide (SO2) before vector delivery enhanced gene transfer efficiency to the nasal epithelium of both mice and rats. SO2 injury also enhanced HIV vector-mediated gene transfer to the tracheas of rodents. These data suggest that SO2 injury increases access of vector to basal cells and/or the basolateral membrane of airway surface epithelial cells. Quantification of gene transfer efficiency in murine tracheas demonstrated that transduction was more efficient when vector was delivered on the day of exposure (7.0%, n = 4) than when vector was delivered on the day after SO2 exposure (1.7%, n = 4).
Analysis of Variance↗
Airway gene transfer in mouse nasal-airways: importance of identification of epithelial type for assessment of gene transfer.
Mouse nasal airways are often used for the assessment of both reporter and cystic fibrosis transmembrane conductance regulator (CFTR) gene transfer to respiratory epithelia. However, the mouse nasal cavity is lined by both olfactory (OE) and respiratory epithelium (RE). Previous gene transfer studies have suggested that OE may be more efficiently transduced by adenoviral vectors than RE. However, to provide data pertinent to CFTR gene transfer in humans, measurements of CFTR function in mice by transepithelial potential difference (TPD) should be directed towards respiratory rather than olfactory epithelium. We report a new technique to mark the position of the TPD sensing cannula tip in the mouse nasal cavity that permitted us to correlate TPD measurements with epithelial cell type. Using this technique, we found TPD values did not discriminate between respiratory and olfactory epithelia. We next assessed relationships between anatomic regions accessed by the TPD cannula and epithelial type. The frequently used insertion depth of approximately 5 mm from the nose tip predominantly recorded the TPD from anterior dorsal olfactory epithelium. Measurement of the TPD of respiratory epithelium in our study was maximized by insertion of the TPD cannula probe to 2.5 mm depth. Because TPD measurements are not sensitive to epithelial type, adequate control of position and TPD catheter insertion depth are required to ensure accurate estimation of CFTR gene transfer into the target RE in the mouse nasal cavity.
Animals↗
Retargeting the coxsackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer.
Lumenal delivery of adenovirus vectors (AdV) results in inefficient gene transfer to human airway epithelium. The human coxsackievirus and adenovirus receptor (hCAR) was detected by immunofluorescence selectively at the basolateral surfaces of freshly excised human airway epithelial cells, suggesting that the absence of apical hCAR constitutes a barrier to adenovirus-mediated gene delivery in vivo. In transfected polarized Madin-Darby canine kidney cells, wild-type hCAR was expressed selectively at the basolateral membrane, whereas hCAR lacking the transmembrane and/or cytoplasmic domains was expressed on both the basolateral and apical membranes. Cells expressing apical hCAR still were not efficiently transduced by AdV applied to the apical surface. However, after the cells were treated with agents that remove components of the apical surface glycocalyx, AdV transduction occurred. These results indicate that adenovirus can infect via receptors located at the apical cell membrane but that the glycocalyx impedes interaction of AdV with apical receptors.
Adenoviruses, Human↗
PKA holoenzyme is functionally coupled to CFTR by AKAPs.
Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferentially regulated by membrane-bound protein kinase A (PKAII). We tested for close physical and functional association of PKA with CFTR in inside-out membrane patches excised from Calu-3 cells. In the presence of MgATP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) increased the product of CFTR channel number and open probability (from 0.36 +/- 0.12 to 1.23 +/- 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular sites by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membrane patches is bound to AKAPs. In whole cell voltage-clamp studies, intracellular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR by extracellular adenosine. These results suggest that AKAPs mediate PKA compartmentalization with CFTR and are required for activation of CFTR by physiological regulators.
Adenosine Triphosphate↗
Intestinal ion transport in NKCC1-deficient mice.
The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) located on the basolateral membrane of intestinal epithelia has been postulated to be the major basolateral Cl(-) entry pathway. With targeted mutagenesis, mice deficient in the NKCC1 protein were generated. The basal short-circuit current did not differ between normal and NKCC1 -/- jejuna. In the -/- jejuna, the forskolin response (22 microA/cm(2); bumetanide insensitive) was significantly attenuated compared with the bumetanide-sensitive response (52 microA/cm(2)) in normal tissue. Ion-replacement studies demonstrated that the forskolin response in the NKCC1 -/- jejuna was HCO(3)(-) dependent, whereas in the normal jejuna it was independent of the HCO(3)(-) concentration in the buffer. NKCC1 -/- ceca exhibited a forskolin response that did not differ significantly from that of normal ceca, but unlike that of normal ceca, was bumetanide insensitive. Ion-substitution studies suggested that basolateral HCO(3)(-) as well as Cl(-) entry (via non-NKCC1) paths played a role in the NKCC1 -/- secretory response. In contrast to cystic fibrosis mice, which lack both basal and stimulated Cl(-) secretion and exhibit severe intestinal pathology, the absence of intestinal pathology in NKCC1 -/- mice likely reflects the ability of the intestine to secrete HCO(3)(-) and Cl(-) by basolateral entry mechanisms independent of NKCC1.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Distribution of ion transport mRNAs throughout murine nose and lung.
Evidence of absorptive or secretory ion transport in different respiratory regions of the mouse was sought by assessing the regional distribution of alpha-, beta-, and gamma-epithelial sodium channel (ENaC; Na(+) absorptive), cystic fibrosis transmembrane conductor regulator (CFTR), and Na(+)-K(+)-2Cl(-) cotransporter mRNAs. High levels of ENaC subunit expression were found in nasal surface epithelium and gland ducts. CFTR was expressed in both superficial nasal respiratory epithelium and glands. These results are consistent with basal amiloride-sensitive Na(+) absorption and cAMP-dependent Cl(-) secretion in murine nasal epithelia. Expression of all three ENaC subunits increased progressively from trachea to terminal bronchioles. Intermediate levels of CFTR and cotransporter expression in bronchial epithelium diminished in bronchioles. The low abundance of CFTR mRNA throughout murine pulmonary epithelium is consistent with functional data that attributes Cl(-) secretion predominantly to an alternative Cl(-) channel. alpha-ENaC as the only mRNA found in all regions of airway epithelia is consistent with the alpha-subunit as requisite for Na(+) absorption, and the increased expression of alpha-, beta-, and gamma-ENaC in distal airways suggests a greater absorptive capability in this region.
Animals↗
Biochemical evidence for an ecto alkaline phosphodiesterase I in human airways.
Because dinucleotides are signaling molecules that can interact with cell surface receptors and regulate the rate of mucociliary clearance in lungs, we studied their metabolism by using human airway epithelial cells. A membrane-bound enzyme was detected on the mucosal surface of polarized epithelia that metabolized dinucleotides with a broad substrate specificity (diadenosine polyphosphates and diuridine polyphosphates [Up(n)U], n = 2 to 6). The enzymatic reaction yielded nucleoside monophosphates (NMP) and Np(n)(-)(1) (N = A or U), and was inhibited by nucleoside 5'-triphosphates (alpha,betamet adenosine triphosphate [ATP] > ATP >/= uridine triphosphate > guanidine triphosphate > cytidine triphosphate). The apparent Michaelis constant (K(m,app)) and apparent maximal velocity (V(max,app)) for [(3)H]Up(4)U were 22 +/- 4 microM and 0.24 +/- 0.05 nmoles. min(-)(1). cm(-)(2), respectively. Thymidine 5'-monophosphate p-nitrophenyl ester and adenosine diphosphate (ADP)- ribose, substrates of ecto alkaline phosphodiesterase I (PDE I) activities, were also hydrolyzed by the apical surface of airway epithelia. ADP-ribose competed with [(3)H]Up(4)U, with a K(i) of 23 +/- 3 microM. The metabolism of ADP-ribose and Ap(4)A was not affected by inhibitors of cyclic nucleotide phosphodiesterases (3-isobutyl-1-methylxanthine, Ro 20-1724, and 1,3-dipropyl-8-p-sulfophenylxanthine), but similarly inhibited by fluoride and N-ethylmaleimide. These results suggest that a PDE I is responsible for the hydrolysis of extracellular dinucleotides in human airways. The wide substrate specificity of PDE I suggests that it may be involved in several signaling events on the luminal surface of airway epithelia, including purinoceptor activation and cell surface protein ribosylation.
Acid Anhydride Hydrolases↗
Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate.
The airway epithelium is resistant to infection by gene transfer vectors when infected from the luminal surface. One strategy for enhancing airway epithelial gene transfer is to modify paracellular permeability, thereby permitting the diffusion of vectors to the basolateral surface, where uptake receptors are expressed. We investigated the ability of a medium-chain fatty acid known to enhance drug absorption, sodium caprate (C10), to increase airway paracellular permeability in comparison with ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Apical application of C10 decreased transepithelial resistance by > 90% within minutes, whereas EGTA required an hour or more to produce a similar effect. C10 increased mannitol and dextran permeability by sevenfold, as compared with a twofold increase produced by EGTA. A greater enhancement of adenoviral lacZ gene transfer was mediated by C10 (50-fold over controls) than by EGTA (10-fold over controls). This correlated with a significant enhancement of adenoviral CFTR-mediated correction of Cl(-) transport in polarized human airway epithelial (HAE) cells from cystic fibrosis (CF) patients. Confocal microscopy revealed a redistribution of claudin-1 following C10 but not EGTA treatment as a possible mechanism of gene-transfer enhancement by C10. These data suggest that C10 may be a better agent for enhancing gene transfer than is EGTA, and that this effect occurs through disruption of claudin-1.
Decanoic Acids↗
Osmotic water permeabilities of cultured, well-differentiated normal and cystic fibrosis airway epithelia.
Current hypotheses describing the function of normal airway surface liquid (ASL) in lung defense are divergent. One theory predicts that normal airways regulate ASL volume by modulating the flow of isosmotic fluid across the epithelium, whereas an alternative theory predicts that ASL is normally hyposmotic. These hypotheses predict different values for the osmotic water permeability (P(f)) of airway epithelia. We measured P(f) of cultures of normal and cystic fibrosis (CF) airway epithelia that, like the native tissue, contain columnar cells facing the lumen and basal cells that face a basement membrane. Xz laser scanning confocal microscopy recorded changes in epithelial height and transepithelial volume flow in response to anisosmotic challenges. With luminal hyperosmotic challenges, transepithelial and apical membrane P(f) are relatively high for both normal and CF airway epithelia, consistent with an isosmotic ASL. Simultaneous measurements of epithelial cell volume and transepithelial water flow revealed that airway columnar epithelial cells behave as osmometers whose volume is controlled by luminal osmolality. Basal cell volume did not change in these experiments. When the serosal side of the epithelium was challenged with hyperosmotic solutions, the basal cells shrank, whereas the lumen-facing columnar cells did not. We conclude that (a) normal and CF airway epithelia have relatively high water permeabilities, consistent with the isosmotic ASL theory, and the capacity to restore water on airway surfaces lost by evaporation, and (b) the columnar cell basolateral membrane and tight junctions limit transepithelial water flow in this tissue.
Bronchi↗
Basal nucleotide levels, release, and metabolism in normal and cystic fibrosis airways.
BACKGROUND: Cystic fibrosis (CF) is a syndrome caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. Despite advances in our understanding of the molecular pathogenesis of CF, the link between CFTR gene mutations and the pathogenesis of CF lung disease remains poorly defined. CFTR has been assigned a number of putative functions that may contribute to innate airway defense, including the regulation of adenosine 5'-triphosphate (ATP) release into the extracellular environment. Because extracellular ATP and uridine 5'-triphosphate (UTP) may regulate airway mucociliary clearance via interaction with luminal P2Y2 receptors, the loss of CFTR-mediated nucleotide release could explain the defect in CF airway defense. MATERIALS AND METHODS: We tested the physiologic importance of CFTR-mediated nucleotide release in vivo by directly measuring levels of ATP and UTP in nasal airway surface liquid from normal and CF subjects. Because these basal nucleotide levels reflect the net activities of nucleotide release and metabolic pathways, we also measured constitutive rates of nucleotide release and metabolism on well-differentiated normal and CF airway cultures in vitro. The measurement of ATP release rates were paralleled by in vivo studies employing continuous nasal perfusion in normal and CF subjects. Finally, the regulation of ATP release by isoproterenol and methacholine-stimulated submucosal gland secretion was tested. RESULTS: These studies revealed that steady-state ATP and UTP levels were similar in normal (470 +/- 131 nM and 37 +/- 7 nM, respectively) and CF (911 +/- 199 nM and 33 +/- 12 nM, respectively) subjects. The rates of both ATP release and metabolism were also similar in normal and CF airway epithelia both in vitro and in vivo. Airway submucosal glands did not secrete nucleotides, but rather, secreted a soluble nucleotidase in response to cholinergic stimuli. CONCLUSION: The concentration of ATP in airway surface liquid is in a range that is relevant for the activation of airway nucleotide receptors. However, despite this finding that suggests endogenous nucleotides may be important for the regulation of mucociliary clearance, our data do not support a role for CFTR in regulating extracellular nucleotide concentrations on airway surfaces.
Adenosine Triphosphate↗