Clobetasol promotes remyelination in a mouse model of neuromyelitis optica.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system that can produce marked neurological deficit. Current NMO therapies include immunosuppressants, plasma exchange and B-cell depletion. Here, we evaluated 14 potential remyelinating drugs emerging from prior small molecule screens done to identify drugs for repurposing in multiple sclerosis and other demyelinating neurological diseases. Compounds were initially evaluated in oligodendrocyte precursor cell (OPC) and cerebellar slice cultures, and then in a mouse model of NMO produced by intracerebral injection of anti-AQP4 autoantibody (AQP4-IgG) and human complement characterized by demyelination with minimal axonal damage. The FDA-approved drug clobetasol promoted differentiation in OPC cultures and remyelination in cerebellar slice cultures and in mice. Intraperitoneal administration of 2 mg/kg/day clobetasol reduced myelin loss by ~60 %, even when clobetasol was administered after demyelination occurred. Clobetasol increased the number of mature oligodendrocytes within lesions without significantly altering initial astrocyte damage or inflammation. These results provide proof-of-concept for the potential utility of a remyelinating approach in the treatment of NMO.

Small molecule screen yields inhibitors of Pseudomonas homoserine lactone-induced host responses.

Pseudomonas aeruginosa infections are commonly associated with cystic fibrosis, pneumonias, neutropenia and burns. The P. aeruginosa quorum sensing molecule N-(3-oxo-dodecanoyl) homoserine lactone (C12) cause multiple deleterious host responses, including repression of NF-κB transcriptional activity and apoptosis. Inhibition of C12-mediated host responses is predicted to reduce P. aeruginosa virulence. We report here a novel, host-targeted approach for potential adjunctive anti-Pseudomonal therapy based on inhibition of C12-mediated host responses. A high-throughput screen was developed to identify C12 inhibitors that restore NF-κB activity in C12-treated, lipopolysaccharide (LPS)-stimulated cells. Triazolo[4,3-a]quinolines with nanomolar potency were identified as C12-inhibitors that restore NF-κB-dependent luciferase expression in LPS- and TNF-stimulated cell lines. In primary macrophages and fibroblasts, triazolo[4,3-a]quinolines inhibited C12 action to restore cytokine secretion in LPS-stimulated cells. Serendipitously, in the absence of an inflammatory stimulus, triazolo[4,3-a]quinolines prevented C12-mediated responses, including cytotoxicity, elevation of cytoplasmic calcium, and p38 MAPK phosphorylation. In vivo efficacy was demonstrated in a murine model of dermal inflammation involving intradermalC12 administration. The discovery of triazolo[4,3-a]quinolines provides a pharmacological tool to investigate C12-mediated host responses, and a potential host-targeted anti-Pseudomonal therapy.

A small molecule screen identifies selective inhibitors of urea transporter UT-A.

Urea transporter (UT) proteins, including UT-A in kidney tubule epithelia and UT-B in vasa recta microvessels, facilitate urinary concentrating function. A screen for UT-A inhibitors was developed in MDCK cells expressing UT-A1, water channel aquaporin-1, and YFP-H148Q/V163S. An inwardly directed urea gradient produces cell shrinking followed by UT-A1-dependent swelling, which was monitored by YFP-H148Q/V163S fluorescence. Screening of ~90,000 synthetic small molecules yielded four classes of UT-A1 inhibitors with low micromolar half-maximal inhibitory concentration that fully and reversibly inhibited urea transport by a noncompetitive mechanism. Structure-activity analysis of >400 analogs revealed UT-A1-selective and UT-A1/UT-B nonselective inhibitors. Docking computations based on homology models of UT-A1 suggested inhibitor binding sites. UT-A inhibitors may be useful as diuretics ("urearetics") with a mechanism of action that may be effective in fluid-retaining conditions in which conventional salt transport-blocking diuretics have limited efficacy.

Microfluidics platform for single-shot dose-response analysis of chloride channel-modulating compounds.

We previously developed cell-based kinetics assays of chloride channel modulators utilizing genetically encoded yellow fluorescent proteins. Fluorescence platereader-based high-throughput screens yielded small-molecule activators and inhibitors of the cAMP-activated chloride channel CFTR and calcium-activated chloride channels, including TMEM16A. Here, we report a microfluidics platform for single-shot determination of concentration-activity relations in which a 1.5 × 1.5 mm square area of adherent cultured cells is exposed for 5-10 min to a pseudo-logarithmic gradient of test compound generated by iterative, two-component channel mixing. Cell fluorescence is imaged following perfusion with an iodide-containing solution to give iodide influx rate at each location in the image field, thus quantifying modulator effects over a wide range of concentrations in a single measurement. IC50 determined for CFTR and TMEM16A activators and inhibitors by single-shot microfluidics were in agreement with conventional plate reader measurements. The microfluidics approach developed here may accelerate the discovery and characterization of chloride channel-targeted drugs.

Fractionation of a herbal antidiarrheal medicine reveals eugenol as an inhibitor of Ca2+-Activated Cl- channel TMEM16A.

The Ca(2+)-activated Cl(-) channel TMEM16A is involved in epithelial fluid secretion, smooth muscle contraction and neurosensory signaling. We identified a Thai herbal antidiarrheal formulation that inhibited TMEM16A Cl(-) conductance. C18-reversed-phase HPLC fractionation of the herbal formulation revealed >98% of TMEM16A inhibition activity in one out of approximately 20 distinct peaks. The purified, active compound was identified as eugenol (4-allyl-2-methoxyphenol), the major component of clove oil. Eugenol fully inhibited TMEM16A Cl(-) conductance with single-site IC(50)~150 µM. Eugenol inhibition of TMEM16A in interstitial cells of Cajal produced strong inhibition of intestinal contraction in mouse ileal segments. TMEM16A Cl(-) channel inhibition adds to the list of eugenol molecular targets and may account for some of its biological activities.

TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells.

TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ∼110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC(50) < 10 µM, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16A(inh)-A01), had an IC(50) of ∼1 µM. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCC(inh)-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16A(inh)-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production.

Inhibition of Ca2+-activated Cl- channels by gallotannins as a possible molecular basis for health benefits of red wine and green tea.

TMEM16A was found recently to be a calcium-activated Cl(-) channel (CaCC). CaCCs perform important functions in cell physiology, including regulation of epithelial secretion, cardiac and neuronal excitability, and smooth muscle contraction. CaCC modulators are of potential utility for treatment of hypertension, diarrhea, and cystic fibrosis. Screening of drug and natural product collections identified tannic acid as an inhibitor of TMEM16A, with IC(50) ∼ 6 µM and ∼100% inhibition at higher concentrations. Tannic acid inhibited CaCCs in multiple cell types but did not affect CFTR Cl(-) channels. Structure-activity analysis indicated the requirement of gallic or digallic acid substituents on a macromolecular scaffold (gallotannins), as are present in green tea and red wine. Other polyphenolic components of teas and wines, including epicatechin, catechin, and malvidin-3-glucoside, poorly inhibited CaCCs. Remarkably, a 1000-fold dilution of red wine and 100-fold dilution of green tea inhibited CaCCs by >50%. Tannic acid, red wine, and green tea inhibited arterial smooth muscle contraction and intestinal Cl(-) secretion. Gallotannins are thus potent CaCC inhibitors whose biological activity provides a potential molecular basis for the cardioprotective and antisecretory benefits of red wine and green tea.

Nanomolar potency pyrimido-pyrrolo-quinoxalinedione CFTR inhibitor reduces cyst size in a polycystic kidney disease model.

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are predicted to slow cyst enlargement in polycystic kidney disease and reduce intestinal fluid loss in secretory diarrheas. Screening of approximately 110000 small synthetic and natural compounds for inhibition of halide influx in CFTR-expressing epithelial cells yielded a new class of pyrimido-pyrrolo-quinoxalinedione (PPQ) CFTR inhibitors. Testing of 347 analogues established structure-activity relationships. The most potent compound, 7,9-dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4',5'-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione, PPQ-102, completely inhibited CFTR chloride current with IC(50) approximately 90 nM. The PPQs, unlike prior CFTR inhibitors, are uncharged at physiological pH, and therefore not subject to membrane potential-dependent cellular partitioning or block efficiency. Patch-clamp analysis confirmed voltage-independent CFTR inhibition by PPQ-102 and showed stabilization of the channel closed state. PPQ-102 prevented cyst expansion and reduced the size of preformed cysts in a neonatal kidney organ culture model of polycystic kidney disease. PPQ-102 is the most potent CFTR inhibitor identified to date.

Thiazolidinone CFTR inhibitors with improved water solubility identified by structure-activity analysis.

The thiazolidinone 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172) inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel conductance with submicromolar affinity and blocks cholera toxin-induced intestinal fluid secretion. Fifty-eight CFTR(inh)-172 analogs were synthesized to identify CFTR inhibitors with improved water solubility, exploring modifications in its two phenyl rings, thiazolidinone core, and core-phenyl connectors. Greatest CFTR inhibition potency was found for 3-CF(3) and polar group-substituted-phenyl rings, and a thiazolidinone core. Two compounds with approximately 1muM CFTR inhibition potency and solubility >180 microM (>10-fold more than CFTR(inh)-172) were identified: Tetrazolo-172, containing 4-tetrazolophenyl in place of 4-carboxyphenyl, and Oxo-172, containing thiazolidinedione in place of the thiazolidinone core. These water soluble thiazolidinone analogs had low cellular toxicity. The improved water solubility of Tetrazolo- and Oxo-172 make them potential lead candidates for therapy of secretory diarrheas and polycystic kidney disease.

Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption.

Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed strongly at the plasma membranes of basal epidermal cells in skin. We found that human skin squamous cell carcinoma strongly overexpresses AQP3. A novel role for AQP3 in skin tumorigenesis was discovered using mice with targeted AQP3 gene disruption. We found that AQP3-null mice were remarkably resistant to the development of skin tumors following exposure to a tumor initiator and phorbol ester promoter. Though tumor initiator challenge produced comparable apoptotic responses in wild-type and AQP3-null mice, promoter-induced cell proliferation was greatly impaired in the AQP3-null epidermis. Reductions of epidermal cell glycerol, its metabolite glycerol-3-phosphate, and ATP were found in AQP3 deficiency without impairment of mitochondrial function. Glycerol supplementation corrected the reduced proliferation and ATP content in AQP3 deficiency, with cellular glycerol, ATP, and proliferative ability being closely correlated. Our data suggest involvement of AQP3-facilitated glycerol transport in epidermal cell proliferation and tumorigenesis by a novel mechanism implicating cellular glycerol as a key determinant of cellular ATP energy. AQP3 may thus be an important determinant in skin tumorigenesis and hence a novel target for tumor prevention and therapy.

Small-molecule screen identifies inhibitors of a human intestinal calcium-activated chloride channel.

Calcium-activated chloride channels (CaCCs) are widely expressed in mammalian tissues, including intestinal epithelia, where they facilitate fluid secretion. Potent, selective CaCC inhibitors have not been available. We established a high-throughput screen for identification of inhibitors of a human intestinal CaCC based on inhibition of ATP/carbachol-stimulated iodide influx in HT-29 cells after lentiviral infection with the yellow fluorescent halide-sensing protein YFP-H148Q/I152L. Screening of 50,000 diverse, drug-like compounds yielded six classes of putative CaCC inhibitors, two of which, 3-acyl-2-aminothiophenes and 5-aryl-2-aminothiazoles, inhibited by >95% iodide influx in HT-29 cells in response to multiple calcium-elevating agonists, including thapsigargin, without inhibition of calcium elevation, calcium-calmodulin kinase II activation, or cystic fibrosis transmembrane conductance regulator chloride channels. These compounds also inhibited calcium-dependent chloride secretion in T84 human intestinal epithelial cells. Patch-clamp analysis indicated inhibition of CaCC gating, which, together with the calcium-calmodulin data, suggests that the inhibitors target the CaCC directly. Structure-activity relationships were established from analysis of more than 1800 analogs, with IC(50) values of the best analogs down to approximately 1 muM. Small-molecule CaCC inhibitors may be useful in pharmacological dissection of CaCC functions and in reducing intestinal fluid losses in CaCC-mediated secretory diarrheas.

Hypertonic saline therapy in cystic fibrosis: Evidence against the proposed mechanism involving aquaporins.

Recent data indicate the clinical benefit of nebulized hypertonic saline in cystic fibrosis lung disease, with a proposed mechanism involving sustained increase in airway surface liquid volume. To account for the paradoxical observation that amiloride suppresses the beneficial effect of hypertonic saline, it has been previously concluded (Donaldson, S. H., Bennett, W. D., Zeman, K. L., Knowles, M. R., Tarran, R., and Boucher, R. C. (2006) N. Engl. J. Med. 354, 241-250) that amiloride-inhibitable aquaporin (AQP) water channels in airway epithelia modulate airway surface liquid volume. Here, we have characterized water permeability and amiloride effects in well differentiated, primary cultures of human airway epithelial cells, stably transfected Fisher rat thyroid epithelial cells expressing individual airway/lung AQPs, and perfused mouse lung. We found high transepithelial water permeability (P(f), 54 +/- 5 microm/s) in airway epithelial cells that was weakly temperature-dependent and inhibited by >90% by reduced pH in the basal membrane-facing solution. Reverse transcription-PCR and immunofluorescence suggested the involvement of AQPs 3, 4, and 5 in high airway water permeability. Experiments using several sensitive measurement methods indicated that amiloride does not inhibit water permeability in non-cystic fibrosis (non-CF) or CF airway epithelia, AQP-transfected Fisher rat thyroid cells, or intact lung. Our data provide evidence against the mechanism proposed by Donaldson et al. to account for the effects of amiloride and hypertonic saline in CF lung disease, indicating the need to identify alternate mechanisms.

Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Cl- channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with Ki < 10 microM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl- conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant Ki from 1.4 microM at +60 mV to 5.6 microM at -60 mV. Apparent potency was reduced by lowering extracellular Cl- concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (-60 mV holding potential, 5 microM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5-8.0, where it exists predominantly as a monovalent anion with solubility approximately 1 mM in water. Topical GlyH-101 (10 microM) in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 microg) reduced by approximately 80% cholera toxin-induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTR(inh)-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of CFTR pore structure, in creating animal models of CF, and as antidiarrheals in enterotoxic-mediated secretory diarrheas.

Increased diffusional mobility of CFTR at the plasma membrane after deletion of its C-terminal PDZ binding motif.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel expressed at the apical plasma membrane. It has been proposed that the C-terminal PDZ binding motif of CFTR is required for its apical membrane targeting and that PDZ-domain interactions may tether CFTR to the actin cytoskeleton via soluble proteins including EBP50/NHERF1 and ezrin. We measured the diffusional mobility of human CFTR in the plasma membrane of Madin-Darby canine kidney cells by photobleaching of green fluorescent protein (GFP)-CFTR chimeras. After bleaching by a focused laser beam, GFP-CFTR fluorescence in the bleached membrane region recovered to approximately 90% of its initial level, indicating that nearly all of the CFTR was mobile. The GFP-CFTR diffusion coefficient (D) was 0.99 +/- 0.09 x 10(-10) cm2/s at 37 degrees C, similar to that of other membrane proteins. GFP-CFTR diffusion was not altered by protein kinase A or C activators but was blocked by paraformaldehyde and filipin. CFTR mutants lacking functional PDZ-binding domains (GFPCFTR-DeltaTRL and GFP-CFTR-DeltaTRA) were also mobile with D significantly increased by approximately 60% compared with GFP-CFTR. However, GFP-CFTR, GFP-CFTR-Delta TRL, and GFP-CFTR-DeltaTRA had similar mobilities (D approximately 12 x 10(-10) cm2/s) at the endoplasmic reticulum in brefeldin A-treated cells. Agents that modulate the actin cytoskeleton (cytochalasin D and jasplakinolide) altered the plasma membrane mobility of CFTR but not CFTR- DeltaTRL. EBP50 (NHERF1), a PDZ domain-containing protein that interacts with the C terminus of CFTR, diffused freely in the cytoplasm with a diffusion coefficient of 0.9 +/- 0.1 x 10(-7) cm2/s. EBP50 diffusion increased by approximately 2-fold after deletion of its ezrin-binding domain. These results indicate that wild-type CFTR is not tethered statically at the plasma membrane but that its diffusion is dependent on PDZ-domain interactions and an intact actin skeleton. PDZ-domain interactions of CFTR are thus dynamic and occur on a time scale of seconds or faster.

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.

Diffusional mobility of the cystic fibrosis transmembrane conductance regulator mutant, delta F508-CFTR, in the endoplasmic reticulum measured by photobleaching of GFP-CFTR chimeras.

Mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis. The most common disease-causing mutation, DeltaF508, is retained in the endoplasmic reticulum (ER) and is unable to function as a plasma membrane chloride channel. To investigate whether the ER retention of DeltaF508-CFTR is caused by immobilization and/or aggregation, we have measured the diffusional mobility of green fluorescent protein (GFP) chimeras of wild type (wt)-CFTR and DeltaF508-CFTR by fluorescence recovery after photobleaching. GFP-labeled DeltaF508-CFTR was localized in the ER and wt-CFTR in the plasma membrane and intracellular membranes in transfected COS7 and Chinese hamster ovary K1 cells. Both chimeras localized to the ER after brefeldin A treatment. Spot photobleaching showed that CFTR diffusion (diffusion coefficient approximately 10(-9) cm(2)/s) was not significantly slowed by the DeltaF508 mutation and that nearly all wt-CFTR and DeltaF508-CFTR diffused throughout the ER without restriction. Stabilization of molecular chaperone interactions by ATP depletion produced remarkable DeltaF508-CFTR immobilization ( approximately 50%) and slowed diffusion (6.5 x 10(-10) cm(2)/s) but had little effect on wt-CFTR. Fluorescence depletion experiments revealed that the immobilized DeltaF508-CFTR in ATP-depleted cells remained in an ER pattern. The mobility of wt-CFTR and DeltaF508-CFTR was reduced by maneuvers that alter CFTR processing or interactions with molecular chaperones, including tunicamycin, geldanamycin, and lactacystin. Photobleaching of the fluorescent ER lipid diOC(4)(3) showed that neither ER restructuring nor fragmentation during these maneuvers was responsible for the slowing and immobilization of CFTR. These results suggest that (a) the ER retention of DeltaF508-CFTR is not due to restricted ER mobility, (b) the majority of DeltaF508-CFTR is not aggregated or bound to slowly moving membrane proteins, and (c) DeltaF508-CFTR may interact to a greater extent with molecular chaperones than does wt-CFTR.

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.

Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists.

Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters.

Defective dietary fat processing in transgenic mice lacking aquaporin-1 water channels.

Immunocytochemistry showed expression of aquaporin-1 (AQP1) water channels at sites involved in dietary fat processing, including intrahepatic cholangiocytes, gallbladder, pancreatic microvascular endothelium, and intestinal lacteals. To determine whether AQP1 has a role in dietary fat digestion and/or absorption, mice were placed on a diet that contained 50% fat. Whereas wild-type mice (3-3.5 wk of age, 10-12 g) gained 49 +/- 5% (SE, n = 50) body weight in 8 days, and heterozygous mice gained 46 +/- 4%, AQP1 null mice gained only 4 +/- 3%; weights became similar after return to a 6% fat diet after 6 days. The null mice on a high-fat diet acquired an oily appearance, developed steatorrhea with increased stool triglyceride content, and manifested serum hypotriglyceridemia. Supplementation of the high-fat diet with pancreatic enzymes partially corrected the decreased weight gain in null mice. Absorption of [(14)C]oleic acid from small intestine was not affected by AQP1 deletion, as determined by blood radioactivity after duodenal infusion. Lipase activity in feces and small intestine was remarkably greater in AQP1 null than wild-type mice on low- and high-fat diets. Fluid collections done in older mice (that are less sensitive to a high-fat diet) by ductal cannulation showed threefold increased pancreatic fluid flow in response to secretin/cholecystokinin, but volumes, pH, and amylase activities were affected little by AQP1 deletion, nor were bile flow rates and bile salt concentrations. Together, these results establish a dietary fat misprocessing defect in AQP1 null mice.

Receptor-mediated targeting of fluorescent probes in living cells.

A strategy was developed to label specified sites in living cells with a wide selection of fluorescent or other probes and applied to study pH regulation in Golgi. cDNA transfection was used to target a single-chain antibody to a specified site such as an organelle lumen. The targeted antibody functioned as a high affinity receptor to trap cell-permeable hapten-fluorophore conjugates. Synthesized conjugates of a hapten (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and fluorescent probes (Bodipy Fl, tetramethylrhodamine, fluorescein) were bound with high affinity (approximately 5 nM) and specific localization to the single-chain antibody expressed in the endoplasmic reticulum, Golgi, and plasma membrane of living Chinese hamster ovary cells. Using the pH-sensitive phOx-fluorescein conjugate and ratio imaging microscopy, pH was measured in the lumen of Golgi (pH 6.25 +/- 0.06). Measurements of pH-dependent vacuolar H+/ATPase pump activity and H+ leak in Golgi provided direct evidence that resting Golgi pH is determined by balanced leak-pump kinetics rather than the inability of the H+/ATPase to pump against an electrochemical gradient. Like expression of the green fluorescent protein, the receptor-mediated fluorophore targeting approach permits specific intracellular fluorescence labeling. A significant advantage of the new approach is the ability to target chemical probes with custom-designed spectral and indicator properties.