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.     

Loss of anion transport without increased sodium absorption characterizes newborn porcine cystic fibrosis airway epithelia

Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR?(/)? pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR?(/)? epithelia showed markedly reduced Cl? and HCO?? transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR?(/)? pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl? conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl? and HCO?? in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.     

Aquaporins and CFTR in Ocular Epithelial Fluid Transport

Aquaporins (AQPs) and the cystic fibrosis transmembrane conductance regulator (CFTR) provide the molecular routes for transport of water and chloride, respectively, through many epithelial tissues. In ocular epithelia, fluid transport generally involves secondary active chloride transport, which creates the osmotic gradient to drive transepithelial water transport. This review is focused on the role of AQPs and CFTR in water and ion transport across corneal/conjunctival epithelia, corneal endothelium, ciliary epithelium, and retinal pigment epithelium. The potential relevance of water and chloride transport to common disorders of ocular fluid balance is also considered. Recent data suggest AQPs and CFTR as attractive targets for drug development for therapy of keratoconjunctivitis sicca, recurrent corneal erosions, corneal edema, glaucoma, retinal detachment, and retinal ischemia.     

Apical CFTR Expression in Human Nasal Epithelium Correlates with Lung Disease in Cystic Fibrosis

Introduction

Although most individuals with cystic fibrosis (CF) develop progressive obstructive lung disease, disease severity is highly variable, even for individuals with similar CFTR mutations. Measurements of chloride transport as expression of CFTR function in nasal epithelial cells correlate with pulmonary function and suggest that F508del-CFTR is expressed at the apical membrane. However, an association between quantitative apical CFTR expression in nasal epithelium and CF disease severity is still missing.

Methods and Materials

Nasal epithelial cells from healthy individuals and individuals with CF between 12–18 years were obtained by nasal brushing. Apical CFTR expression was measured by confocal microscopy using CFTR mAb 596. Expression was compared between both groups and expression in CF nasal epithelial cells was associated with standardized pulmonary function (FEV₁%).

Results

The proportion of cells expressing apical CFTR in columnar epithelium is lower in CF compared to non-CF. The apical CFTR expression level was significantly correlated with FEV₁% in F508del homozygous subjects (r = 0.63, p = 0.012).

Conclusion

CFTR expression in nasal epithelial cells is lower in subjects with CF compared to healthy subjects. The proportion of cells expressing F508del-CFTR at the apical membrane is variable between subjects and is positively correlated with FEV₁% in F508del-CFTR homozygous subjects.     

Trichomonas vaginalis infection impairs anion secretion in vaginal epithelium

Trichomonas vaginalis is a common protozoan parasite, which causes trichomoniasis associated with severe adverse reproductive outcomes. However, the underlying pathogenesis has not been fully understood. As the first line of defense against invading pathogens, the vaginal epithelial cells are highly responsive to environmental stimuli and contribute to the formation of the optimal luminal fluid microenvironment. The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel widely distributed at the apical membrane of epithelial cells, plays a crucial role in mediating the secretion of Cl⁻ and HCO₃⁻. In this study, we investigated the effect of T. vaginalis on vaginal epithelial ion transport elicited by prostaglandin E₂ (PGE₂), a major prostaglandin in the semen. Luminal administration of PGE₂ triggered a remarkable and sustained increase of short-circuit current (I_SC) in rat vaginal epithelium, which was mainly due to Cl⁻ and HCO₃⁻ secretion mediated by the cAMP-activated CFTR. However, T. vaginalis infection significantly abrogated the I_SC response evoked by PGE₂, indicating impaired transepithelial anion transport via CFTR. Using a primary cell culture system of rat vaginal epithelium and a human vaginal epithelial cell line, we demonstrated that the expression of CFTR was significantly down-regulated after T. vaginalis infection. In addition, defective Cl⁻ transport function of CFTR was observed in T. vaginalis-infected cells by measuring intracellular Cl⁻ signals. Conclusively, T. vaginalis restrained exogenous PGE₂-induced anion secretion through down-regulation of CFTR in vaginal epithelium. These results provide novel insights into the intervention of reproductive complications associated with T. vaginalis infection such as infertility and disequilibrium in vaginal fluid microenvironment.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

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.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

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.     

A primary culture model of differentiated murine tracheal epithelium

The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances ( approximately 12 kOmega. cm(2)) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and alpha-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine beta-defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.     

Cytoskeleton and CFTR

Cystic Fibrosis Transmembrane conductance Regulator, CFTR, is a membrane protein expressed in epithelia. A protein kinase A (PKA)-regulated Cl⁻ channel, it is a rate-limiting factor in fluid transport. Mutations in CFTR are responsible for cystic fibrosis, CF, an autosomal recessive disease. The most frequent mutation is deletion of phenylalanine at position 508, ΔF508. The regulation of trafficking and degradation of CFTR/ΔF508CFTR as well as its function(s) is a complex process which involves a number of proteins including chaperones and adaptors. It is now known that cytoskeletal proteins, previously considered only as structural proteins, are also important factors in the regulation of cellular processes and functions. The aim of the present review is to focus on how microfilaments, microtubules and intermediary filaments form a dynamic interactome with CFTR to participate in the regulation of CFTR-dependent transepithelial ion transport, CFTR trafficking and degradation.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance

Introduction

We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl⁻) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.

Methods

Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR^−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl⁻ transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca²⁺]_i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.

Results

Sinupret-mediated Cl⁻ secretion [ΔI_SC(µA/cm²)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR^−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca²⁺ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl⁻ secretion. Sinupret also enhanced CBF and ASL depth.

Conclusion

Sinupret stimulates CBF, promotes transepithelial Cl⁻ secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca²⁺-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl⁻ secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.     

Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR

Gene transfer of CFTR cDNA to airway epithelia is a promising approach to treat cystic fibrosis (CF). Most gene transfer vectors use strong viral promoters even though the endogenous CFTR promoter is very weak. To learn whether expressing CFTR at a low level in a fraction of cells would correct Cl(-) transport, we mixed freshly isolated wild-type and CF airway epithelial cells in varying proportions and generated differentiated epithelia. Epithelia with approximately 20% wild-type cells generated approximately 70% the transepithelial Cl(-) current of epithelia containing 100% wild-type cells. These data were nearly identical to those previously obtained with CFTR expressed under control of a strong promoter in a CF epithelial cell line. We also tested high level CFTR expression using the very strong cytomegalovirus (CMV) promoter as well as the cytokeratin-18 (K18) promoter. In differentiated airway epithelia, the CMV promoter generated 50-fold more transgene expression than the K18 promoter, but the K18 promoter generated more transepithelial Cl(-) current at high vector doses. Using functional studies, we found that with marked overexpression, some CFTR channels were present in the basolateral membrane where they shunted Cl(-) flow, thereby reducing net transepithelial Cl(-) transport. These results suggest that very little CFTR is required in a fraction of CF epithelial cells to complement Cl(-) transport because transepithelial Cl(-) flow is limited at the basolateral membrane. Thus they suggest a broad leeway in promoter strength for correcting the CF gene transfer, although at very high expression levels CFTR may be mislocalized to the basolateral membrane.     

Murine nasal septa for respiratory epithelial air-liquid interface cultures

Air-liquid interface models using murine tracheal respiratory epithelium have revolutionized the in vitro study of pulmonary diseases. This model is often impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. We describe a simple technique to harvest the murine nasal septum and grow the epithelial cells in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their respective cultured epithelium at an air-liquid interface were compared by scanning electron microscopy (SEM). Immunocytochemistry for type IV beta-tubulin and zona occludens-1 (Zo-1) are performed to determine differentiation and confluence, respectively. To rule out contamination with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein (OMP) was performed. Transepithelial resistance and potential measurements were determined using a modified vertical Ussing chamber SEM reveals approximately 90% ciliated respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and potential confirming a confluent monolayer with electrically active airway epitheliumn containing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is a superior source when compared with the trachea. The nasal septum increases the yield of respiratory epithelial cells up to 8-fold.     

Recovery of ΔF508-CFTR function by analogs of hyaluronan disaccharide

We recently discovered that hyaluronan was exported from fibroblasts by MRP5 and from epithelial cells by cystic fibrosis (CF) transmembrane conductance regulator (CFTR) that was known as a chloride channel. On this basis we developed membrane permeable analogs of hyaluronan disaccharide as new class of compounds to modify their efflux. We found substances that activated hyaluronan export from human breast cancer cells. The most active compound 2-(2-acetamido-3,5-dihydroxyphenoxy)-5-aminobenzoic acid (Hylout4) was tested for its influence on the activity of epithelial cells. It activated the ion efflux by normal and defective ΔF508-CFTR. It also enhanced the plasma membrane concentration of the ΔF508-CFTR protein and reduced the transepithelial resistance of epithelial cells. In human trials of healthy persons, it caused an opening of CFTR in the nasal epithelium. Thus compound Hylout4 is a corrector that recovered ΔF508-CFTR from intracellular degradation and activated its export function.     

Correlation of results of immunocytochemical, electrophysiologic, and molecular studies in patients with cystic fibrosis

In 13 cystic fibrosis (CF) patients of 5 to 23 years of age with a known mutation spectrum of gene CFTR, sweat chloride values and nasal-potential differences (NPD) were measured and localization characteristics of the protein product of gene CFTR in the cells of nasal epithelium were studied. Sweat Chloride values were normal or boundary (24 to 62 mM/l) in six CF patients. In seven CF patients, these values were significantly above the estimates for the control group. On average, the NPD values were -44.7 +/- 2.2 mV (from -32.5 to -68.9 mV) and -17.2 +/- 1.8 mV (from -6.8 to -30.2 mV) in CF patients and the control group, respectively. Histochemical studies clearly revealed the localization of the CFTR protein on the apical membrane of the nasal epithelium. Depending on the type of mutation, the protein product of gene CFTR was either absent or regularly distributed in the cytoplasm in CF patients; it was not detected in the apical membrane. Thus, NPD measurements and the analysis of the localization of the protein product of gene CFTR in scrapes of nasal epithelium were shown to be additional, highly informative methods of CF diagnostics.     

Airway epithelial wounds in rhesus monkey generate ionic currents that guide cell migration to promote healing

Damage to the respiratory epithelium is one of the most critical steps to many life-threatening diseases, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. The mechanisms underlying repair of the damaged epithelium have not yet been fully elucidated. Here we provide experimental evidence suggesting a novel mechanism for wound repair: endogenous electric currents. It is known that the airway epithelium maintains a voltage difference referred to as the transepithelial potential. Using a noninvasive vibrating probe, we demonstrate that wounds in the epithelium of trachea from rhesus monkeys generate significant outward electric currents. A small slit wound produced an outward current (1.59 μA/cm(2)), which could be enhanced (nearly doubled) by the ion transport stimulator aminophylline. In addition, inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) with CFTR(Inh)-172 significantly reduced wound currents (0.17 μA/cm(2)), implicating an important role of ion transporters in wound induced electric potentials. Time-lapse video microscopy showed that applied electric fields (EFs) induced robust directional migration of primary tracheobronchial epithelial cells from rhesus monkeys, towards the cathode, with a threshold of <23 mV/mm. Reversal of the field polarity induced cell migration towards the new cathode. We further demonstrate that application of an EF promoted wound healing in a monolayer wound healing assay. Our results suggest that endogenous electric currents at sites of tracheal epithelial injury may direct cell migration, which could benefit restitution of damaged airway mucosa. Manipulation of ion transport may lead to novel therapeutic approaches to repair damaged respiratory epithelium.     

Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.     

Airway Surface Liquid Volume Regulation Determines Different Airway Phenotypes in Liddle Compared with βENaC-overexpressing Mice

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na⁺ channel β-subunit (βENaC-Tg) suggest that raised airway Na⁺ transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function βENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, βENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na⁺ transport measured in Ussing chambers (“flooded” conditions) was raised in both Liddle and βENaC-Tg mice. Because enhanced Na⁺ transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic “thin film” conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na⁺ absorption were intact in Liddle but defective in βENaC-Tg mice. We conclude that the capacity to regulate Na⁺ transport and ASL volume, not absolute Na⁺ transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.     

CFTR, MDR1, and MRP1 immunolocalization in normal human nasal respiratory mucosa.

CFTR (cystic fibrosis transmembrane conductance regulator), MDR1 (multidrug resistance), and MRP1 (multidrug resistance-associated protein), members of the ABC transporter superfamily, possess multiple functions, particularly Cl(-), anion, and glutathione conjugate transport and cell detoxification. They are also hypothesized to have a number of complementary functions. It is generally accepted that data obtained from nasal mucosa can be extrapolated to lower airway cell physiology. The aim of the present study was to investigate by immunohistochemistry the differential localization of CFTR, MDR1, and MRP1 in the normal mucosa of 10 human nasal turbinates. In ciliated epithelial cells, CFTR was inconstantly expressed at the apical cell surface, intense membranous labeling was observed for MDR1, and intense cytoplasmic labeling was observed for MRP1. In the glands, a higher level of expression was observed on serous cells, at the apical surface (for CFTR), on lateral membranes (for MDR1), and with an intracytoplasmic distribution (for MRP1). In conclusion, CFTR, MDR1 and MRP1 are expressed in the epithelium and glands of the nasal respiratory mucosa, but with different patterns of expression. These results suggest major roles for CFTR, MDR1, and MRP1 in serous glandular cells and a protective function for MDR1 and MRP1 in respiratory ciliated cells. (J Histochem Cytochem 48:1215-1222, 2000)     

Characterization of Nasal Potential Difference in cftr Knockout and F508del-CFTR Mice

Background

Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR) defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF). Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V_TE) to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V_TE in CF mice must be well characterized for correct interpretation.

Methods

We performed V_TE measurements in large-scale studies of two mouse models of CF—B6;129 cftr knockout and FVB F508del-CFTR—and their respective wild-type (WT) littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice.

Results

We determined the typical V_TE values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl^- solution was considered to indicate a normal response.

Conclusions

These data will make it possible to interpret changes in nasal V_TE in mouse models of CF, in future preclinical studies.