[Ion transporters in the lungs. Use as therapeutic targets]. / Transportadores de iones en pulmón. Uso como dianas terapéuticas.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

Transportadores de iones en pulmón: Uso como dianas terapéuticas / Ion transporters in the lungs. Use as therapeutic targets

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

Genistein antagonizes gliadin-induced CFTR malfunction in models of celiac disease.

In celiac disease (CD), an intolerance to dietary gluten/gliadin, antigenic gliadin peptides trigger an HLA-DQ2/DQ8-restricted adaptive Th1 immune response. Epithelial stress, induced by other non-antigenic gliadin peptides, is required for gliadin to become fully immunogenic. We found that cystic-fibrosis-transmembrane-conductance-regulator (CFTR) acts as membrane receptor for gliadin-derived peptide P31-43, as it binds to CFTR and impairs its channel function. P31-43-induced CFTR malfunction generates epithelial stress and intestinal inflammation. Maintaining CFTR in an active open conformation by the CFTR potentiators VX-770 (Ivacaftor) or Vrx-532, prevents P31-43 binding to CFTR and controls gliadin-induced manifestations. Here, we evaluated the possibility that the over-the-counter nutraceutical genistein, known to potentiate CFTR function, would allow to control gliadin-induced alterations. We demonstrated that pre-treatment with genistein prevented P31-43-induced CFTR malfunction and an epithelial stress response in Caco-2 cells. These effects were abrogated when the CFTR gene was knocked out by CRISP/Cas9 technology, indicating that genistein protects intestinal epithelial cells by potentiating CFTR function. Notably, genistein protected gliadin-sensitive mice from intestinal CFTR malfunction and gliadin-induced inflammation as it prevented gliadin-induced IFN-γ production by celiac peripheral-blood-mononuclear-cells (PBMC) cultured ex-vivo in the presence of P31-43-challenged Caco-2 cells. Our results indicate that natural compounds capable to increase CFTR channel gating might be used for the treatment of CD.

Potentiators exert distinct effects on human, murine, and Xenopus CFTR.

VX-770 (Ivacaftor) has been approved for clinical usage in cystic fibrosis patients with several CFTR mutations. Yet the binding site(s) on CFTR for this compound and other small molecule potentiators are unknown. We hypothesize that insight into this question could be gained by comparing the effect of potentiators on CFTR channels from different origins, e.g., human, mouse, and Xenopus (frog). In the present study, we combined this comparative molecular pharmacology approach with that of computer-aided drug discovery to identify and characterize new potentiators of CFTR and to explore possible mechanism of action. Our results demonstrate that 1) VX-770, NPPB, GlyH-101, P1, P2, and P3 all exhibited ortholog-specific behavior in that they potentiated hCFTR, mCFTR, and xCFTR with different efficacies; 2) P1, P2, and P3 potentiated hCFTR in excised macropatches in a manner dependent on the degree of PKA-mediated stimulation; 3) P1 and P2 did not have additive effects, suggesting that these compounds might share binding sites. Also 4) using a pharmacophore modeling approach, we identified three new potentiators (IOWH-032, OSSK-2, and OSSK-3) that have structures similar to GlyH-101 and that also exhibit ortholog-specific potentiation of CFTR. These could potentially serve as lead compounds for development of new drugs for the treatment of cystic fibrosis. The ortholog-specific behavior of these compounds suggest that a comparative pharmacology approach, using cross-ortholog chimeras, may be useful for identification of binding sites on human CFTR.

The novel dry extract BNO 1011 stimulates chloride transport and ciliary beat frequency in human respiratory epithelial cultures.

BACKGROUND: Herbal remedies predate written history and continue to be used more frequently than conventional pharmaceutical medications. The novel dry extract BNO 1011 is based on a combination of five herbs that is used to treat acute and chronic rhinosinusitis. We evaluated the pharmacologic effects of the novel dry extract BNO 1011 on human respiratory epithelial cultures specifically addressing electrolyte transport and cilia beat frequency (CBF). METHODS: Well-differentiated human bronchial epithelial cultures grown at an air-liquid interface were treated on the apical or basolateral surface with varying concentrations of dry extract BNO 1011. Changes in transepithelial sodium and chloride transport were determined in Ussing chambers under voltage-clamped conditions. Changes in CBF were determined using the Sissons-Ammons Video Analysis system (Ammons Engineering, Mt. Morris, MI). RESULTS: When applied to the apical surface, dry extract BNO 1011 activated forskolin-stimulated chloride secretion and ciliary beat in a dose-dependent fashion. Basolateral application of dry extract BNO 1011 did not alter the measured physiological properties. CONCLUSION: Apical application of dry extract BNO 1011 stimulates both chloride secretion and CBF and therefore may augment mucociliary clearance.

Curcumin and genistein additively potentiate G551D-CFTR.

BACKGROUND: The G551D mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is a common cause of cystic fibrosis (CF). G551D-CFTR is characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Numerous small molecules have been shown to increase the activity of G551D-CFTR presumably by binding to the CFTR protein. METHODS: We investigated the effect of curcumin, genistein and their combined application on G551D-CFTR activity using the patch clamp technique. RESULTS: Curcumin increased G551D-CFTR whole-cell and single-channel currents less than genistein did at their maximally effective concentrations. However, curcumin further increased the channel activity of G551D-CFTR that had been already maximally potentiated by genistein, up to ~50% of the WT-CFTR level. In addition, the combined application of genistein and curcumin over a lower concentration range synergistically rescued the gating defect of G551D-CFTR. CONCLUSIONS: The additive effects between curcumin and genistein not only support the hypothesis that multiple mechanisms are involved in the action of CFTR potentiators, but also pose pharmaceutical implications in the development of drugs for CF pharmacotherapy.

Functional regulation of cystic fibrosis transmembrane conductance regulator-containing macromolecular complexes: a small-molecule inhibitor approach.

CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein-protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)-NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR-NHERF2-LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2-NHERF2 interaction. We show that this compound can disrupt the LPA2-NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl- channel function can be finely tuned by modulating PDZ domain-based protein-protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl- channels.

The possible additional role of the cystic fibrosis transmembrane regulator to motoneuron inhibition produced by glycine effects.

In the present work we study the contribution of the chloride channel of the Cystic Fibrosis Transmembrane Regulator (CFTR) in the postsynaptic inhibition of somatic motoneurons during rapid-eye-movement (REM) sleep atonia. Postsynaptic inhibition of motoneurons is partially responsible for the atonia that occurs during REM sleep. Disfacilitation is an additional mechanism that lowers motoneuron excitability in this state. Postsynaptic inhibition is mediated by the release of glycine from synaptic terminals on motoneurons, and by GABA that plays a complementary role to that of glycine. In this work we look in brain stem motoneurons of neonatal rats at a mechanism unrelated to the actions of glycine, GABA or to disfacilitation which depends on the chloride channel of the CFTR. We studied the presence of CFTR by immunocytochemistry. In electrophysiological experiments utilizing whole cell recordings in in vitro slices we examined the consequences of blocking this chloride channel. The effects on motoneurons of the application of glycine, of the application of glibenclamide (a CFTR blocker) and again of glycine during the effects of glibenclamide were studied. Glycine produced an hyperpolarization, a decrease in motoneuron excitability and a decrease in input resistance, all characteristic changes of the postsynaptic inhibition produced by this neurotransmitter. Glibenclamide produced an increase in input resistance and in motoneurons' repetitive discharge as well as a shift in the equilibrium potential for chloride ions as indicated by the displacement of the reversal potential for glycinergic actions. In motoneurons treated with glibenclamide, glycine produced postsynaptic inhibition but this effect was smaller when compared to that elicited by glycine in control conditions. The fact that blocking of the CFTR-chloride channel in brain stem motoneurons influences glycinergic inhibition suggests that this channel may play a complementary role in the glycinergic inhibition that occurs during REM sleep.

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator.

Proteomic analysis has proved to be an important tool for understanding the complex nature of genetic disorders, such as cystic fibrosis (CF), by defining the cellular protein environment (proteome) associated with wild-type and mutant proteins. Proteomic screens identified the proteome of CF transmembrane conductance regulator (CFTR), and provided fundamental information to studies designed for understanding the crucial components of physiological CFTR function. Simultaneously, high-throughput screens for small-molecular correctors of CFTR mutants provided promising candidates for therapy. The majority of CF cases are caused by nucleotide deletions (DeltaF508 CFTR; >75%), resulting in CFTR misfolding, or insertion of premature termination codons ( approximately 10%), leading to unstable mRNA and reduced levels of truncated dysfunctional CFTR. In this article, we review recent results of proteomic screens, developments in identifying correctors for the most frequent CFTR mutants, and comment on how integration of the knowledge gained from these studies may aid in finding a cure for CF and a number of other genetic disorders.

Substance P stimulates human airway submucosal gland secretion mainly via a CFTR-dependent process.

Chronic bacterial airway infections are the major cause of mortality in cystic fibrosis (CF). Normal airway defenses include reflex stimulation of submucosal gland mucus secretion by sensory neurons that release substance P (SubP). CFTR is an anion channel involved in fluid secretion and mutated in CF; the role of CFTR in secretions stimulated by SubP is unknown. We used optical methods to measure SubP-mediated secretion from human submucosal glands in lung transplant tissue. Glands from control but not CF subjects responded to mucosal chili oil. Similarly, serosal SubP stimulated secretion in more than 60% of control glands but only 4% of CF glands. Secretion triggered by SubP was synergistic with vasoactive intestinal peptide and/or forskolin but not with carbachol; synergy was absent in CF glands. Pig glands demonstrated a nearly 10-fold greater response to SubP. In 10 of 11 control glands isolated by fine dissection, SubP caused cell volume loss, lumen expansion, and mucus flow, but in 3 of 4 CF glands, it induced lumen narrowing. Thus, in CF, the reduced ability of mucosal irritants to stimulate airway gland secretion via SubP may be another factor that predisposes the airways to infections.

Effects of Cordyceps sinensis, Cordyceps militaris and their isolated compounds on ion transport in Calu-3 human airway epithelial cells.

AIM OF THE STUDY: The traditional Chinese medicine Cordyceps sinensis (CS) (Clavicipitaceae) improves pulmonary function and is used to treat respiratory disease. Here, we compare the efficacy and mechanisms of action of Cordyceps sinensis and Cordyceps militaris (CM) (Clavicipitaceae) in Calu-3 human airway epithelial monolayer model. MATERIAL AND METHODS: The extracts of Cordyceps sinensis and Cordyceps militaris, as well as their isolated compounds, cordycepin and adenosine, stimulated ion transport in a dose-dependent manner in Calu-3 monolayers. In subsequent experiments, transport inhibitor bumetanide and carbonic anhydrase inhibitor acetazolamide were added after Cordyceps sinensis and Cordyceps militaris extracts to determine their effects on Cl- and HCO3- movement. RESULTS: The results suggested that Cordyceps sinensis and Cordyceps militaris extracts may affect the anion movement from the basolateral to apical compartments in the airway epithelia. CONCLUSIONS: Basolateral Na+-K+-2Cl- cotransporter and apical cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel are involved in the process. The results provide the first evidence for the pharmacological mechanism of Cordyceps sinensis and Cordyceps militaris on respiratory tract.

Dependence of cAMP meditated increases in Cl- and HCO(3)- permeability on CFTR in bovine corneal endothelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is present on the apical membrane of corneal endothelial cells. Increasing intracellular [cAMP] with forskolin stimulates an NPPB and glibenclamide-inhibitable apical Cl(-) and HCO(3)(-) permeability [Sun, X.C., Bonanno, J.A., 2002. Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells. Am. J. Physiol. Cell Physiol. 282, C673-C683]. To definitively determine that the increased permeability is dependent on CFTR, we used an siRNA knockdown approach. Apical Cl(-) and HCO(3)(-) permeability and steady-state HCO(3)(-) flux were measured in the presence or absence of forskolin using cultured bovine corneal endothelial cells that were transfected with CFTR siRNA or a scrambled sequence control. CFTR protein expression was reduced by approximately 80% in CFTR siRNA treated cultures. Forskolin (10 microM) increased apical chloride permeability by 7-fold, which was reduced to control level in siRNA treated cells. CFTR siRNA treatment had no effect on baseline apical chloride permeability. Apical HCO(3)(-) permeability was increased 2-fold by 10 microM forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO(3)(-) permeability by knocking down CFTR expression. The steady-state apical-basolateral pH gradient (DeltapH) at 4h in control cultures was increased approximately 2.5-fold by forskolin. In CFTR siRNA treated cells, the baseline DeltapH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO(3)(-) permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO(3)(-) permeability.

4-Chlorobenzo[F]isoquinoline (CBIQ), a novel activator of CFTR and DeltaF508 CFTR.

4-Chlorobenzo[F]isoquinoline (CBIQ) is a novel compound, here shown to activate both CFTR (cystic fibrosis transmembrane conductance regulator) Cl- ion channels and KCNN4, intermediate conductance, calcium-sensitive K+-channels, present in transporting epithelia by the use of heterologous expression systems. Earlier studies with other benzoquinolines, namely 7,8- and 5,6 benzoquinoline, showed they too could activate CFTR and KCNN4, but the evidence was only indirect. However this study also shows that CBIQ can also activate DeltaF508 CFTR, the most common mutant form of CFTR present in approximately 75% of patients with cystic fibrosis. This property is not shared with the other benzoquinolines. As activation of CFTR and KCNN4 work in unison to promote epithelial chloride secretion, CBIQ is a new chemical scaffold for developing agents that may be useful in cystic fibrosis.

Cortisol receptor blockade and seawater adaptation in the euryhaline teleost Fundulus heteroclitus.

To examine the role of cortisol in seawater osmoregulation in a euryhaline teleost, adult killifish were acclimated to brackish water (10 per thousand) and RU486 or vehicle was administered orally in peanut oil daily for five days at low (40 mg.kg(-1)) or high dose (200 mg.kg(-1)). Fish were transferred to 1.5 x seawater (45 per thousand) or to brackish water (control) and sampled at 24 h and 48 h after transfer, when Cl- secretion is upregulated. At 24 h, opercular membrane Cl- secretion rate, as Isc, was increased only in the high dose RU486 group. Stimulation of membranes by 3-isobutyl-1-methylxanthine and cAMP increased Isc in vehicle treated controls but those from RU486-treated animals were unchanged and membranes from brackish water animals showed a decrease in Isc. At 48 h, Isc increased and transepithelial resistance decreased in vehicle and RU486 groups, compared to brackish water controls. Plasma cortisol increased in all groups transferred to high salinity, compared to brackish water controls. RU486 treated animals had higher cortisol levels compared to vehicle controls. Vehicle treated controls had lower cortisol levels than untreated or RU486 treated animals, higher stimulation of Isc, and lower hematocrit at 24 h, beneficial effects attributed to increased caloric intake from the peanut oil vehicle. Chloride cell density was significantly increased in the high dose RU486 group at 48 hours, yet Isc was unchanged, suggesting a decrease in Cl- secretion per cell. Thus cortisol enhances NaCl secretion capacity in chloride cells, likely via glucocorticoid type receptors.

High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl(-) conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 microm together with a low concentration of forskolin (0.5 microm) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl(-) sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl(-) currents rapidly (<5 min) with K(d) down to 200 nm and were CFTR-selective, reversible, and nontoxic. Several compounds, the most potent being a trifluoromethylphenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (K(d) > 10 microm). When added for 10 min, none of the compounds activated DeltaPhe(508)-CFTR in transfected cells grown at 37 degrees C (with DeltaPhe(508)-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48 h of growth at 27 degrees C, tetrahydrocarbazol and N-phenyltriazine derivatives strongly stimulated Cl(-) conductance with K(d) < 1 microm. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.

Novel role for CFTR in fluid absorption from the distal airspaces of the lung.

The active absorption of fluid from the airspaces of the lung is important for the resolution of clinical pulmonary edema. Although ENaC channels provide a major route for Na(+) absorption, the route of Cl(-) transport has been unclear. We applied a series of complementary approaches to define the role of Cl(-) transport in fluid clearance in the distal airspaces of the intact mouse lung, using wild-type and cystic fibrosis Delta F508 mice. Initial studies in wild-type mice showed marked inhibition of fluid clearance by Cl(-) channel inhibitors and Cl(-) ion substitution, providing evidence for a transcellular route for Cl(-) transport. In response to cAMP stimulation by isoproterenol, clearance was inhibited by the CFTR inhibitor glibenclamide in both wild-type mice and the normal human lung. Although isoproterenol markedly increased fluid absorption in wild-type mice, there was no effect in Delta F508 mice. Radioisotopic clearance studies done at 23 degrees C (to block active fluid absorption) showed approximately 20% clearance of (22)Na in 30 min both without and with isoproterenol. However, the clearance of (36)Cl was increased by 47% by isoproterenol in wild-type mice but was not changed in Delta F508 mice, providing independent evidence for involvement of CFTR in cAMP-stimulated Cl(-) transport. Further, CFTR played a major role in fluid clearance in a mouse model of acute volume-overload pulmonary edema. After infusion of saline (40% body weight), the lung wet-to-dry weight ratio increased by 28% in wild-type versus 64% in Delta F508 mice. These results provide direct evidence for a functionally important role for CFTR in the distal airspaces of the lung.