Intestinal current measurements (ICM) from rectal biopsies are a sensitive means to detect cystic fibrosis transmembrane conductance regulator (CFTR) function, but have not been optimized for multicenter use. We piloted multicenter standard operating procedures (SOPs) to detect CFTR activity by ICM and examined key questions for use in clinical trials. SOPs for ICM using human rectal biopsies were developed across three centers and used to characterize ion transport from non-CF and CF subjects (two severe CFTR mutations). All data were centrally evaluated by a blinded interpreter. SOPs were then used across four centers to examine the effect of cold storage on CFTR currents and compare CFTR currents in biopsies from one subject studied simultaneously either at two sites (24 hours post-biopsy) or when biopsies were obtained by either forceps or suction. Rectal biopsies from 44 non-CF and 17 CF subjects were analyzed. Mean differences (µA/cm2; 95% confidence intervals) between CF and non-CF were forskolin/IBMX=102.6(128.0 to 81.1), carbachol=96.3(118.7 to 73.9), forskolin/IBMX+carbachol=200.9(243.1 to 158.6), and bumetanide=-44.6 (-33.7 to -55.6) (P<0.005, CF vs non-CF for all parameters). Receiver Operating Characteristic curves indicated that each parameter discriminated CF from non-CF subjects (area under the curve of 0.94-0.98). CFTR dependent currents following 18-24 hours of cold storage for forskolin/IBMX, carbachol, and forskolin/IBMX+carbachol stimulation (n=17 non-CF subjects) were 44%, 47.5%, and 47.3%, respectively of those in fresh biopsies. CFTR-dependent currents from biopsies studied after cold storage at two sites simultaneously demonstrated moderate correlation (n=14 non-CF subjects, Pearson correlation coefficients 0.389, 0.484, and 0.533). Similar CFTR dependent currents were detected from fresh biopsies obtained by either forceps or suction (within-subject comparisons, n=22 biopsies from three non-CF subjects). Multicenter ICM is a feasible CFTR outcome measure that discriminates CF from non-CF ion transport, offers unique advantages over other CFTR bioassays, and warrants further development as a potential CFTR biomarker.

Transport of chloride through the cystic fibrosis transmembrane conductance regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates [1, 2]. Loss of CFTR function leads to cystic fibrosis [1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine, and vas deferens. Conversely, uncontrolled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera [5, 6] and other secretory diarrheas [7] as well as polycystic kidney disease [8-10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation, and characterization of a mutation in the zebrafish cse1I gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the uncontrolled activation of the CFTR channel. Analyses in zebrafish larvae and mammalian cells indicate that Cse1I is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much-needed model system to study CFTR regulation in vivo.

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca2+-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl− secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca2+, suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.

Anion transport by the colonic mucosa maintains the hydration and pH of the colonic lumen, and its disruption causes a variety of diarrheal diseases. Cholinergic agonists raise cytosolic Ca2+ levels and stimulate anion secretion, but the mechanisms underlying this effect remain unclear. Cholinergic stimulation of anion secretion may occur via activation of Ca2+-activated Cl- channels (CaCCs) or an increase in the Cl- driving force through CFTR after activation of Ca2+-dependent K+ channels. Here we investigated the role of a candidate CaCC protein, bestrophin-2 (Best2), using Best2(-/-) mice. Cholinergic stimulation of anion current was greatly reduced in Best2(-/-) mice, consistent with our proposed role for Best2 as a CaCC. However, immunostaining revealed Best2 localized to the basolateral membrane of mucin-secreting colonic goblet cells, not the apical membrane of Cl--secreting enterocytes. In addition, in the absence of HCO3-, cholinergic-activated current was identical in control and Best2(-/-) tissue preparations, which suggests that most of the Best2 current was carried by HCO3-. These data delineate an alternative model of cholinergic regulation of colonic anion secretion in which goblet cells play a critical role in HCO3- homeostasis. We therefore propose that Best2 is a HCO3- channel that works in concert with a Cl:HCO3- exchanger in the apical membrane to affect transcellular HCO3- transport. Furthermore, previous models implicating CFTR in cholinergic Cl- secretion may be explained by substantial downregulation of Best2 in Cftr(-/-) mice.

Progressive lung damage in cystic fibrosis (CF) has been linked to inadequate airway mucosal hydration. We previously demonstrated that an inositol tetrakisphosphate analog, 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl)ester (INO-4995), regulates airway secretory and absorptive processes, affecting mucosal hydration by prolonged (24 h) inhibition of Na(+) and fluid absorption in CF human nasal epithelia (CFHNE). The objectives of this study were to further assess clinical potential of INO-4995 in CF through ascertaining in vivo activity in mice with CF, determining the effects of repeated administration on potency and determining cytoplasmic half-life. Uptake and metabolism of [(3)H]INO-4995 was monitored with HPLC to calculate intracellular half-life. INO-4995 was administered in vitro repeatedly over 4 to 8 days to CFHNE. Fluid absorption was assessed by blue dextran exclusion, and basal short-circuit current was measured in Ussing chambers. INO-4995 (1-100 microg/kg) was dosed intranasally either as a single dose or once per day over 4 days to gut-corrected CF mice. [(3)H]INO-4995 was rapidly taken up by epithelial cultures and converted to the active drug, which had a half-life of 40 hours. Repeated daily application of INO-4995 to CFHNE lowered the effective concentration for inhibition of fluid absorption and amiloride-sensitive short-circuit current in cultured CFHNE, and reduced nasal potential difference to nearly control levels in gut-corrected CF mice. Ca(2+)-activated Cl(-) channel activity was also boosted in cultures. Mouse nasal levels fell from abnormal levels to within 2 muA of normal with repeated exposure to 0.8 microg/kg over 4 days. These data support further development of INO-4995 for the treatment of CF.

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl(-) and Na(+) epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%-65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.

For almost two decades, it has been postulated that calcium-activated Cl– channels (CaCCs) play a role in airway epithelial Cl– secretion, but until recently, the molecular identity of the airway CaCC(s) was unknown. Recent studies have unequivocally identified TMEM16A as a glandular epithelial CaCC. We have studied the airway bioelectrics of neonatal mice homozygous for a null allele of Tmem16a (Tmem16a–/–) to investigate the role of this channel in Cl– secretion in airway surface epithelium. When compared with wild-type tracheas, the Tmem16a–/– tracheas exhibited a >60% reduction in purinoceptor (UTP)-regulated CaCC activity. Other members of the Tmem16 gene family, including Tmem16f and Tmem16k, were also detected by reverse transcription-PCR in neonatal tracheal epithelium, suggesting that other family members could be considered as contributing to the small residual UTP response. TMEM16A, however, appeared to contribute little to unstimulated Cl– secretion, whereas studies with cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice and wild-type littermates revealed that unstimulated Cl– secretion reflected ∼50% CFTR activity and ∼50% non-Tmem16a activity. Interestingly, the tracheas of both the Tmem16a–/– and the CFTR–/– mice exhibited similar congenital cartilaginous defects that may reflect a common Cl– secretory defect mediated by the molecularly distinct Cl– channels. Importantly, the residual CaCC activity in Tmem16a–/– mice appeared inadequate for normal airway hydration because Tmem16a–/– tracheas exhibited significant, neonatal, lumenal mucus accumulation. Our data suggest that TMEM16A CaCC-mediated Cl– secretion appears to be necessary for normal airway surface liquid homeostasis.

The efficiency of the mucociliary clearance (MCC) process that removes noxious materials from airway surfaces depends on the balance between mucin secretion, airway surface liquid (ASL) volume, and ciliary beating. Effective mucin dispersion into ASL requires salt and water secretion onto the mucosal surface, but how mucin secretion rate is coordinated with ion and, ultimately, water transport rates is poorly understood. Several components of MCC, including electrolyte and water transport, are regulated by nucleotides in the ASL interacting with purinergic receptors. Using polarized monolayers of airway epithelial Calu-3 cells, we investigated whether mucin secretion was accompanied by nucleotide release. Electron microscopic analyses of Calu-3 cells identified subapical granules that resembled goblet cell mucin granules. Real-time confocal microscopic analyses revealed that subapical granules, labelled with FM 1-43 or quinacrine, were competent for Ca(2+)-regulated exocytosis. Granules containing MUC5AC were apically secreted via Ca(2+)-regulated exocytosis as demonstrated by combined immunolocalization and slot blot analyses. In addition, Calu-3 cells exhibited Ca(2+)-regulated apical release of ATP and UDP-glucose, a substrate of glycosylation reactions within the secretory pathway. Neither mucin secretion nor ATP release from Calu-3 cells were affected by activation or inhibition of the cystic fibrosis transmembrane conductance regulator. In SPOC1 cells, an airway goblet cell model, purinergic P2Y(2) receptor-stimulated increase of cytosolic Ca(2+) concentration resulted in secretion of both mucins and nucleotides. Our data suggest that nucleotide release is a mechanism by which mucin-secreting goblet cells produce paracrine signals for mucin hydration within the ASL.

Amiloride-sensitive, epithelial Na(+) channel (ENaC)-mediated, active absorption of Na(+) is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na(+) and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and (22)Na(+) flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives.

The airway epithelium occupies a critical environmental interface, protecting the host from a wide variety of inhaled insults, including chemical and particulate pollutants and pathogens. The coordinated regulation of ion and water transport, mucous secretion, and cilia beating underlies mucociliary clearance. Physical trapping and removal of harmful substances, in combination with baseline or inducible secretion of antimicrobial factors, antioxidants, and protease inhibitors and recruitment of nonspecific inflammatory cells (neutrophils, monocytes), constitutes airway innate host defense.

Immunofluorescence labelling and confocal microscopy were employed to examine the polarized distribution of several membrane transport proteins believed to be essential for salivary secretion in the rat submandibular gland. The Na+/K+-ATPase, Na+/H+ exchanger isoform 1 (NHE1), and the secretory Na+/K+/2Cl–cotransporter isoform were all found in the basolateral membranes of acinar and intralobular duct cells. Anion exchanger isoform 2 (AE2) was found only in the basolateral membranes of acinar cells, while AE1 was absent from glandular epithelial cells. Aquaporin 5 was detected in the apical membranes of acinar cells, while the cystic fibrosis transmembrane conductance regulator was found only in apical membranes of intralobular duct cells. NHEs 2 and 3 were found in the apical membranes of both acinar and intralobular duct cells. Our results are generally consistent with the expected distribution of most transporters based on previous physiological and pharmacological experiments. However, the apical localization of NHEs 2 and 3, and the presence of the secretory isoform of the Na+/K+/2Cl–cotransporter in intralobular duct cells were not predicted.