INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THE MOST PREVALENT LETHAL genetic disease, cystic fibrosis (CF), is produced by mutations in the CF transmembrane conductance regulator (CFTR) protein. Normally, Cl-permeable epithelial cells in airways, pancreas, and other tissues are Cl impermeable in CF, resulting in lung infection, pancreatic insufficiency, and other disease manifestations. The most common CF-causing CFTR mutation, F508, is present in at least one allele in >90% of CF patients (17). F508 CFTR appears to be retained in the endoplasmic reticulum of target epithelial cells, where it is functional but mildly misfolded (16, 17). Low temperature (5), chemical chaperones (3, 19), and other agents (18, 24) can correct the CFTR misprocessing to yield Cl-permeable cells. It is believed that restoration of 5-10% of CFTR-mediated Cl permeability in CF cells may be of substantial therapeutic value in CF (13, 17). An important goal in CF therapy is thus the identification of compounds that restore Cl permeability in CF epithelial cells.

The discovery of novel compounds to correct defective CFTR function and/or cellular processing requires a sensitive quantitative assay of CFTR-mediated halide transport suitable for high-throughput screening. We previously introduced quinolinium (15) and pyrido[2,1-h]-pteridin (12) chemical halide indicators for functional CFTR measurements in cell culture models. Although suitable in principle for high-throughput screening, chemical-based halide indicators require cell loading and washing and have imperfect cell retention. Recently, we introduced a green fluorescent protein (GFP)-based halide indicator that could serve as an intracellular Cl/I sensor (11). The GFP-based indicator does not require cell loading or washing, is retained perfectly within cells, and has very good optical properties and photostability.

The purpose of this study was to establish the technical details, experimental conditions, and cell lines to measure CFTR-mediated halide permeability efficiently in cells grown on 96-well plates and assayed in a commercial fluorescence plate reader. The requirements for cell lines included epithelial origin, bright cytoplasmic expression of yellow fluorescent protein (YFP), stable expression of wild-type or mutant CFTRs, low basal (cAMP-independent) halide permeability, and robust growth on plastic 96-well plates. In addition, the formation of electrically tight monolayers is useful for secondary analysis by short-circuit current measurements. We characterize here the sensitivity of the plate reader assay and demonstrate its utility for screening of CFTR activators. In a separate study (8), the methods developed here were applied to identify novel CFTR activators from a combinatorial compound library containing analogs of flavone and benzoquinolizinium CFTR activators.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cell culture and transfections. Swiss 3T3 fibroblasts that stably express wild-type CFTR were provided by Dr. Michael Welsh. Fischer rat thyroid (FRT) cells were transfected with wild-type or G551D CFTR and selected in 0.6 mg/ml zeocin. After stable clones were generated, cells were transiently or stably transfected with YFP-H148Q using Lipofectamine (GIBCO BRL) for fibroblasts or Effectene (Qiagen) for FRT cells according to the manufacturer's instructions. For transient transfection, cells were seeded on 18-mm-diameter round glass coverslips and transfected after 24 h with 1 µg of plasmid pcDNA3.1 containing the humanized YFP-H148Q coding sequence. For stable expression, CFTR-expressing 3T3 and FRT cells were selected with 0.2 mg of hygromycin B or 0.75 mg/ml G418, respectively, and clonal populations were obtained by repeated limiting dilution and cloning rings. The stable cell lines could be passed at least 15 times without a decrease in YFP-H148Q fluorescence. In addition, a stably transfected Chinese hamster ovary (CHO) cell line was generated that expressed YFP-H148Q and was subsequently transfected with an episomal plasmid (pCEP4) containing the coding sequence for wild-type or G551D CFTR. CHO cells expressing YFP-H148Q and CFTR were selected in 0.5 mg/ml G418 and hygromycin B. C127 cells expressing wild-type or F508 CFTR (provided by Marko Pregel, Genzyme) were transiently transfected with the plasmid encoding YFP-H148Q.

Fluorescence microscopy. Cells plated on coverslips were mounted in a low-volume chamber and perfused with PBS (in mM: 137 NaCl, 2.7 KCl, 0.7 CaCl₂, 1.1 MgCl₂, 1.5 KH₂PO₄, and 8.1 Na₂HPO₄, pH 7.4) at 8-10 ml/min at 37°C. Cell fluorescence was measured continuously on an inverted epifluorescence microscope (Nikon Diaphot) using a dry objective (Leitz, ×25, numerical aperture 0.35), YFP filter set (500 ± 10 nm excitation, 535 ± 15 nm emission, 515 nm dichroic), and photomultiplier detector. For Cl/I exchange, cells were perfused with modified PBS in which a specified amount of NaCl was replaced with NaI.

Intracellular pH measurements. 3T3 cells expressing wild-type CFTR were cultured on glass coverslips and incubated with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)- AM (20 µM) in PBS for 10 min. After the cells were washed, they were mounted in the perfusion chamber and positioned on the microscope stage. Cell fluorescence was monitored at 520 ± 15 at 440- and 490-nm excitation wavelengths. Cells were subjected to the same series of solution additions as used to measure CFTR activity. At the end of the experiment, a pH calibration was performed using perfusion solutions containing ionophores (20 µM nigericin, 20 µM valinomycin) and high K⁺ (in mM: 137 KCl, 2 NaCl, 1 CaCl₂, 0.5 MgCl₂, 10 HEPES, and 10 MES) with specified pH in the range 6.5-7.5.

Short-circuit current measurements. FRT cells stably expressing wild-type or G551D CFTR were cultured on Snapwell inserts (Costar) for 8-10 days to form epithelial monolayers with high electrical resistance (3-4 k · cm²). The inserts were mounted in an Ussing chamber system (vertical diffusion chamber, Costar) in which the apical chamber was filled with 5 ml of a physiological solution containing 65 mM NaCl and 65 mM sodium gluconate. The basolateral chamber contained 130 mM NaCl as the main salt, and the basolateral membrane was permeabilized with 250 µg/ml amphotericin B. The transepithelial potential was clamped at zero using a DVC-1000 voltage clamp (World Precision Instruments) via Ag-AgCl electrodes and 1 M KCl agar bridges, and short-circuit current was recorded by a 16-bit analog-to-digital converter (PC-516 DAQ board, National Instruments) using a collection/display program written using LabView software (National Instruments).

Fluorescence plate reader assay. Cells stably expressing CFTR (wild type or G551D) and YFP-H148Q were plated in 96-well black microplates (Corning Costar) at a density of 20,000 cells/well using a Labsystems Multidrop apparatus for automated liquid delivery. After 24-72 h, the cells were washed three times with 200 µl of PBS using a Labsystems Cellwash apparatus and incubated at 37°C for 30 min. The residual volume of PBS was 40 µl/well. In some experiments this fluid was replaced before the assay with an equal volume of PBS containing specified concentrations of putative CFTR activators. The assay was performed in a FLUOstar Galaxy microplate reader (BMG LabTechnologies) equipped with HQ500/20X (500 ± 10 nm) excitation and HQ535/30M (535 ± 15 nm) emission filters (Chroma Technology) and two syringe pumps. YFP fluorescence was monitored continuously for 100 s for each well, and data were binned in 0.5-s intervals. At 5 s after the start of fluorescence recording, 110 µl of a modified PBS (137 mM NaI replacing NaCl) were delivered by a syringe pump to give an extracellular I concentration of 100 mM. After an additional 15 s, a second syringe pump delivered 50 µl of the PBS/NaI buffer containing forskolin. Assays were performed at 37°C unless otherwise indicated.

Data analysis. Data are shown as representative curves or as averages (±SE) of fitted results. Where necessary, background measured in the absence of cells was subtracted. The rate of fluorescence change was quantified from the maximal slope using an exponential regression.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We previously reported that the fluorescence of the GFP mutant YFP-H148Q is sensitive to pH and halides, with a substantially greater decrease in fluorescence produced by I than Cl (12). The feasibility of detecting CFTR activation was demonstrated in a microscopy assay using CFTR-expressing Swiss 3T3 fibroblasts that were transiently transfected with YFP-H148Q. After incubating cells in buffer containing 100 mM I for 30 min, extracellular I was replaced by Cl and then forskolin was added to activate CFTR. Figure 1A (top trace) shows a similar assay in 3T3 cells expressing wild-type CFTR and a modified YFP-H148Q in which cell expression was increased approximately fivefold by engineering an improved Kozak's sequence and employing eukaryotic codon usage. YFP fluorescence changed little after replacement of I by Cl and then increased promptly after addition of forskolin to the Cl-containing solution. Although a clear increase in fluorescence after CFTR stimulation was found, the problem with this assay for quantitative high-throughput screening is the need to preincubate cells with an I-containing buffer, which can affect cell viability and adhesion, stimulate basal halide permeability, and establish an indeterminant electrochemical driving force because of uncertain intracellular I concentration. Furthermore, for high-throughput screening using 96-well microplates, cells in different wells would be exposed to I for different amounts of time, resulting in different electrochemical driving forces.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Fluorescence microscopy assay of cystic fibrosis transmembrane conductance regulator (CFTR) function in cells expressing YFP-H148Q. A, top trace: time course of cell fluorescence in 3T3 fibroblasts expressing wild-type CFTR. Cells were incubated in 100 mM I, and then I was replaced by Cl, followed by addition of 5 µM forskolin as indicated. Middle trace: similar study, except that I was added to cells that were incubated with physiological Cl. Bottom trace: Same experiments done in control 3T3 fibroblasts not expressing CFTR. B: experiments as in A done in Fischer rat thyroid (FRT) cells expressing G551D CFTR. Where indicated, forskolin in the absence or presence of 100 µM genistein was added. C: C127 cells expressing F508 CFTR were cultured at physiological or low temperature before measurements. See text for details. YFP, yellow fluorescent protein.

The detection of I rather than Cl influx provides a theoretical advantage because anion cotransporters such as NKCC and exchangers such as AE1 transport I poorly, whereas CFTR transports I efficiently. Figure 1A (middle trace) shows a modified assay in which the halide exchange is done in the reverse sequence. Cells bathed in a physiological Cl-containing solution are subjected to an inwardly directed I gradient in which 100 mM Cl is replaced by I. YFP fluorescence in cells expressing wild-type CFTR decreased slowly before forskolin addition, as a result of basal halide permeability, and then rapidly after forskolin addition. The prompt fluorescence increase on reperfusion with the high-Cl buffer occurs because CFTR is fully activated. The same experiment in CFTR-null cells showed similar basal halide permeability but no effect of forskolin (Fig. 1A, bottom trace). This simplified assay does not require preincubation of cells with an I-containing solution and thus eliminates the concerns mentioned above.

Figure 1, B and C, demonstrates the utility of the assay to characterize the function of CFTR mutants. Activation of G551D CFTR, a mutant that is processed normally but is relatively Cl impermeable at the plasma membrane (17), was studied in stably transfected FRT cells. Halide transport was activated by a combination of genistein and forskolin, whereas forskolin alone had little effect. Figure 1C shows that the assay can detect the temperature-dependent correction of F508 CFTR misprocessing in transfected C127 cells after incubation at 27°C for 48 h. No cAMP-dependent halide transport was detected when the cells were maintained at 37°C.

A potential concern with the YFP-based assay is that YFP fluorescence is sensitive to changes in both halide concentration and pH. This is an unavoidable concern because the halide-sensing mechanism of YFP involves a shift in pK_a (11). To quantify the contribution of pH changes to the observed changes in YFP fluorescence, cytoplasmic pH was measured during the CFTR activation protocol using BCECF as an intracellular pH indicator. Figure 2A shows the time course of cytoplasmic pH in response to forskolin activation of wild-type CFTR in 3T3 cells expressing wild-type CFTR but not YFP-H148Q. A small change of <0.15 pH units was found. To determine the change in YFP fluorescence that would be produced by the observed pH change, pH/I titrations of purified recombinant YFP-H148Q protein were done (Fig. 2B). A 0.15-unit change in pH produced a small change in apparent I sensitivity, indicating that pH changes occurring during the assay could account for at most <10% of the change in YFP fluorescence on addition of agonist.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Influence of pH change on the assay of CFTR function. A: time course of cytoplasmic pH, measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence, in cells expressing wild-type CFTR subjected to Cl/I exchange/forskolin addition protocol as in Fig. 1. Where indicated, the perfusate contained nigericin, valinomycin, and high K+ (pH 7.5 or 6.5) to calibrate absolute pH. B: I sensitivity of purified recombinant YFP-H148Q at pH 7.2 () and pH 7.35 ().

Several cell lines were screened for their utility in rapid CFTR screening. The characteristics that were evaluated included growth on uncoated 96-well plastic plates, expression of YFP-H148Q after transient transfection, and basal halide permeability. Low basal halide permeability (before addition of forskolin or other agonists) is an important requirement, because the sensitivity of the YFP-based gain-of-function assay depends on detecting an increase in downward slope of the fluorescence vs. time curve. 3T3 fibroblasts expressing CFTR were evaluated because they show strong cAMP-dependent Cl transport and are very bright after transfection with YFP-H148Q. However, they required collagen coating of the plate to avoid detachment during washing and solution additions in the plate reader. FRT and CHO cells do not require collagen coating and were found to be resistant to the washing and solution dispensing procedures. 3T3, FRT, and CHO were transfected with YFP-H148Q to generate stable cell lines to be used in the plate reader assay. Cells appeared bright with a signal that was 4- to 10-fold greater than background in the fluorescence plate reader, with CHO cells being the brightest.

Several maneuvers were tested in an attempt to decrease basal halide permeability, including changes in assay temperature, use of mildly hyperosmolar assay medium (medium + mannitol, final 325 mosM), or addition of indomethacin (20 µM for 30 min before the assay), bumetanide, furosemide, and DIDS (100 µM each). The rationale for the use of hyperosmolar medium was to prevent activation of volume-activated Cl channels. The rationale for the use of the inhibitors was to prevent activation of calcium-activated Cl channels (DIDS) and to block prostaglandin production (indomethacin) and halide cotransport (bumetanide, furosemide, DIDS). Representative data for FRT cells expressing wild-type CFTR are shown in Fig. 3. At room temperature, cells showed a relatively high forskolin-independent I influx. Significant reduction of this I leak was found by incubating the cells for 5 min at 37°C and carrying out the assay at 37°C. The inhibitors and hyperosmolar medium were tested under these conditions. DIDS, indomethacin, and hyperosmolar medium were without significant effect. Bumetanide and furosemide produced a small but significant reduction in halide transport. The greatest effect was found by incubation of cells for 1 h at 37°C. Carrying out the assay at 37°C (instead of at room temperature, 22-24°C) consistently reduced basal halide permeability in most cell types and is the preferred temperature to mimic in vivo cell physiology.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Basal (unstimulated) halide permeability in FRT cells. Cells expressing CFTR and YFP-H148Q were exposed to 100 mM I, and the cell fluorescence was measured using the plate reader. Representative curves are shown in A, and averaged results (means ± SE, n = 3-5 cultures) are shown in B. Assays were done at 23°C or 37°C, in which cells were incubated at 37°C for 5 min or 1 h before assays. Where indicated, solutions contained furosemide, bumetanide, DIDS, indomethacin, or mannitol (hyperosmolality). See text for details. *P < 0.05; **P < 0.01.

To perform CFTR functional assays in a commercial plate reader, we generated stably transfected cell lines expressing wild-type or mutant CFTR together with YFP-H148Q. Cells were plated in black-wall, clear-bottom 96-well microplates. The volumes, rates, and hardware for solution additions were optimized to eliminate artifactual fluorescence signals during continuous recordings from individual wells. Utilizing two software-controlled syringe pumps, the assay involved addition of an I-containing solution to generate a 100 mM inwardly directed I gradient, followed by addition of forskolin without changing the extracellular I concentration. Figure 4A (left) shows the response to forskolin (5 µM) in CHO, 3T3, and FRT cells stably expressing wild-type CFTR. The fluorescence signal consisted of an initial slow decrease due to basal I permeability followed by a more rapid decrease in fluorescence resulting from CFTR activation. As expected, only slow halide transport was seen in CFTR null cells even after forskolin addition. Figure 4A (right) gives a representative fluorescence micrograph of the plated cells showing bright and uniform cell-to-cell fluorescence. High-magnification confocal microscopy showed a fairly uniform intracellular distribution in cytoplasm and nucleus (not shown). Figure 4B shows representative data in FRT cells expressing wild-type CFTR as a function of forskolin concentration, in which the forskolin was added 10 min before (left) or during (right) the assay. Significant activation of halide transport was seen at forskolin concentrations as low as 125 nM. The data were quite reproducible from well to well, as shown by the 8 superimposed curves taken at random from a 96-well plate in which each well was stimulated by 5 µM forskolin (Fig. 4B, right, bottom trace). The full plate mean and standard deviation for the downward slope (see METHODS) was 0.0082 ± 0.0008 s¹. The delay in response to forskolin added during the assay is probably due to the time required for forskolin to cross the plasma membrane and activate adenylyl cyclase to increase intracellular cAMP, since no delay was seen when cells were prestimulated by forskolin (Fig. 4B, left).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Forskolin stimulation of CFTR in YFP-H148Q-expressing cells cultured on 96-well plates and assayed using the fluorescence plate reader. A, left: representative time courses of fluorescence in Chinese hamster ovary (CHO), 3T3, and FRT cells expressing wild-type CFTR, and CHO cells without CFTR. Assays done as in Fig. 1 using 5 µM forskolin. Right: fluorescence micrograph of FRT cells expressing wild-type CFTR and YFP-H148Q. B, left: time course of fluorescence in FRT cells expressing wild-type CFTR in which indicated concentrations of forskolin were added 5 min before the assay. Right: forskolin added during the assay as indicated. Curves obtained with 5 µM forskolin from 8 random wells of a 96-well plate are superimposed. C, left: dose-response relationship for the experiment shown in B, right. The ordinate is the maximal slope of the fluorescence curve after forskolin addition. Right: short-circuit current (Isc) analysis of CFTR activation in FRT cells expressing wild-type CFTR in response to forskolin addition. See text for further explanation.

Figure 4C (left) summarizes the dose-response relation for activation of wild-type CFTR by forskolin in the stably transfected FRT cells. To quantify the sensitivity of the fluorescence plate reader assay, we measured short-circuit current in FRT monolayers in response to different concentrations of forskolin. From Ussing chamber short-circuit measurements, 125 nM forskolin, the lowest concentration producing a significant fluorescence change in the plate reader assay, gave a short-circuit current equal to 2.2 ± 0.4% of the maximum current measured at 5 µM forskolin (Fig. 4C). These results establish the sensitivity and reproducibility of the plate reader fluorescence assay.

We evaluated with the plate reader assay the efficacy of genistein, a known activator of CFTR (8, 21, 23), to induce halide transport in cells expressing wild-type and G551D CFTR. Genistein was added to wells at different concentrations before the assay to mimic a high-throughput screening procedure in which putative CFTR activators are tested. Forskolin was added during the assay at the submaximal concentration of 250 nM for wild-type CFTR or the high concentration of 5 µM for G551D CFTR. Figure 5A shows that genistein produced a dose-dependent activation of wild-type CFTR. Representative curves are shown on the left, and the averaged dose-response data on the right. At the highest genistein concentration (12.5-50 µM), there was a rapid fluorescence decrease in response to the initial I addition. At lower genistein concentrations there was no increase in basal halide transport (direct CFTR activation), but the activation of CFTR by forskolin was potentiated. In FRT cells stably expressing G551D CFTR, genistein alone did not activate halide transport, but subsequent addition of forskolin gave a small but significant activation of G551D CFTR at the higher genistein concentrations (Fig. 5B).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Genistein-mediated activation of halide transport. A: FRT cells expressing wild-type CFTR were pretreated with the indicated concentrations of genistein and assayed in the plate reader using 100 mM I and 250 nM forskolin. Left: original fluorescence time course data. Right: averaged dose-response relationship. B: same as in A, except on FRT cells expressing G551D CFTR.

In addition to genistein, a number of other compounds have been reported to activate wild-type CFTR (reviewed in Ref. 20). We systematically compared the efficacy of these reported CFTR activators in the plate reader assay. The compounds genistein, apigenin, 8- cyclopentyl-1,3-dipropylxanthine (CPX), 3-isobutyl-1-methylxanthine (IBMX), chlorzoxazone, milrinone, bromotetramisole, levamisole, and 8-methoxypsoralen (8-MPO) were tested at 5, 25, and 50 µM concentrations. Representative experiments from single wells in Fig. 6A (left) show weak activation of CFTR by 8-MPO and strong activation by genistein, apigenin, and IBMX (all 50 µM).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Activators of CFTR and alternative Cl channels. A, left: fluorescence plate reader assays were done in FRT cells expressing wild-type CFTR after preincubation with indicated compounds (50 µM). Right: activation of non-CFTR Cl channels by stimulation with 100 µM ATP or 1 µM ionomycin. Cells were CHO transfected with YFP-H148Q. B: averaged dose-response data (mean ± SE, n = 3 cultures) for cells incubated with 5, 25, and 50 µM concentrations of indicated compounds. 8-MPO, 8-methoxypsoralen; CPX, 8-cyclopentyl-1,3-dipropylxanthine.

Averaged dose-response data are summarized in Fig. 6B. Under identical assay conditions, IBMX was the most potent CFTR activator followed by apigenin and genistein. 8-MPO, CPX, and milrinone were relatively weak CFTR activators. Chlorzoxazone, bromotetramisole, and levamisole were ineffective, and the latter two tested at 1 mM as well (not shown). Activation of alternative Cl channels was also tested using the compounds ATP (100 µM) and ionomycin (1 µM), which produce an increase in intracellular Ca²⁺ concentration in CHO cells (10) and consequent activation of Ca²⁺-dependent Cl channels. Figure 6A (right) shows activation of alternative Cl channels in CHO cells that do not express CFTR.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The purpose of this study was to establish a practical method for quantitative high-throughput screening of potential modulators of CFTR halide permeability. The assay utilizes a genetically targeted fluorescent YFP indicator to give a real-time readout of intracellular halide concentration. In addition to its excellent optical properties and retention in cells, the YFP indicator is particularly advantageous for high-throughput screening because the time-consuming and potentially destructive steps of indicator loading and washing are not necessary. In the I/Cl exchange protocol developed here, CFTR function is assayed in cells grown on 96-well microplates without any intervention between cell plating and assay, except for addition of test compounds or other maneuvers (e.g., growth at low temperature, transfection with CFTR-interacting proteins) as desired. After having established the indicator and assay conditions, the major challenge was the generation of cells lines that stably expressed CFTRs together with the YFP indicator: cell lines with minimal basal halide permeability before cAMP stimulation and cell lines that grow well on uncoated plastic 96-well plates. A set of cell lines was selected that had already been used successfully in studies of CFTR function, such as 3T3 fibroblasts, FRT cells, and CHO cells. The cell lines were screened for robust growth on plastic, bright, and uniform YFP expression, and, most importantly, low basal halide permeability. 3T3 fibroblasts required collagen coating and are thus suitable for small-scale studies but not for high-throughput screening. FRT and CHO cells were quite robust and suitable for measurement of CFTR activity. FRT cells were preferred because they are from an epithelial cell line that forms electrically tight junctions and permits efficient secondary assay of cell Cl conductance by short-circuit current analysis.

Various maneuvers including assay temperature, hyperosmolality, and transport inhibitors were tested to minimize basal halide permeability. Neither Ca²⁺-dependent nor volume-sensitive Cl channels appeared to contribute to basal halide transport in the assay, nor did prostaglandin-mediated activation. Bumetanide and furosemide significantly decreased halide transport, suggesting a small contribution from a halide cotransporter. Cell incubation and assay at 37°C, which markedly reduced cAMP-independent halide leak in multiple cell types, was used for subsequent screening studies.

The stable cell lines generated here were suitable for CFTR functional assays using a conventional fluorescence plate reader equipped with syringe pumps and temperature control. The assay was able to detect small increases in CFTR activity by the agonists forskolin and genistein. The sensitivity of a cell-based assay is an important consideration in a drug discovery project, since relatively low potency compounds may be encountered in the initial screen of a combinatorial drug library. The sensitivity of the YFP plate reader assay for detection of CFTR activators in the transfected FRT cells was estimated by comparison of fluorescence plate reader data with short-circuit current measurements. Taking as 100% activity the stimulation of wild-type CFTR by maximal (5 µM) forskolin, the plate reader assay could detect with significance an increase in CFTR halide transport in response to 125 nM forskolin, which gives in short-circuit current measurements a Cl current ~2% of maximum. In initial high-throughput screens it should be possible to identify new chemical classes of CFTR activators with relatively low potency.

A number of compounds have been found to activate CFTR in a variety of cell types and using different assays and experimental conditions (20). Some studies have compared the efficacy of flavonoids and xanthines (4, 9). However, there is in general a lack of comparative studies testing different classes of compounds using the same assay. It is important to quantify the relative efficacy of CFTR activators for the design and synthesis of lead-based combinatorial libraries. We tested the activity of a series of putative CFTR agonists at concentrations up to 50 µM. IBMX was the strongest activator, probably because of its ability to inhibit the phosphodiesterase and increase intracellular cAMP (14). Apigenin and genistein, flavone-type CFTR activators (9), were also able to induce a strong response, whereas milrinone (14), CPX (1), and 8-MPO (6) were weaker activators. Chlrozoxazone (21), bromotetramisole, and levamisole (2) did not activate CFTR in our assay, which is consistent with a report that they may induce Cl secretion indirectly by activating basolateral membrane K⁺ channels (21). The lack of effect of levamisole and bromotetramisole, which probably act by inhibition of cell phosphatases (2), may be a cell-specific phenomenon. Genistein and apigenin were potent activators of CFTR, which mechanistically have been proposed to activate CFTR by direct CFTR binding (22, 23).

Given the limited number of steps that are required, our assay is readily adapted to automated screening using integrated robotic systems. The assay can identify at the same time compounds that activate CFTR directly, potentiate the effect of forskolin, or function as CFTR inhibitors. For the identification of acute CFTR activators or inhibitors, the cells are washed with PBS, incubated at 37°C to reduce basal transport, treated with the tested compounds, and assayed in the plate reader. For the identification of compounds that might correct the F508 CFTR trafficking defect, test compounds are added to cells for 12-48 h before the assay. Cells are then washed and assayed in the plate reader. Further improvements in the assay sensitivity are in progress, including the identification of mutated YFPs with ultra-high halide sensitivities (7).