CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts

Hervé Barrière, Chantal Poujeol, Michel Tauc, Jean Michel Blasi, Laurent Counillon, and Philippe Poujeol

Unité Mixte de Recherche-Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To study the potential influence of cystic fibrosis conductance regulator (CFTR) on intracellular pH regulation during apoptosis induction, we used PS120 Chinese hamster lung fibroblasts devoid of the Na+/H+ exchanger (NHE1 isoform) transfected with constructs, allowing the expression of CFTR and/or NHE1. Kinetics of lovastatin-induced apoptosis were measured by orcein staining, double staining with Hoechst-33258, propidium iodide, DNA fragmentation, and annexin V labeling. In PS120 control cells, the percentage of apoptotic cells after 40 h of lovastatin treatment was 23 ± 3%, whereas in PS120 CFTR-transfected cells, this percentage was 40 ± 4%. In PS120 NHE1 cells, the transfection with CFTR did not modify the percentage of apoptotic cells after 40 h (control: 19 ± 3%, n = 8; CFTR: 17 ± 1%, n = 8), indicating that blocking intracellular acidification by overexpressing the Na+/H+ exchanger inhibited the enhancement of apoptosis induced by CFTR. In all cell lines, the initial pH values were identical (pH = 7.46 ± 0.04, n = 9), and treatment with lovastatin led to intracellular acidification. However, the pH value after 40 h was lower in PS120 CFTR-transfected cells (pH = 6.85 ± 0.02, n = 10) than in PS120 cells (pH = 7.15 ± 0.03, n = 10). To further investigate the origin of this increased intracellular acidification observed in CFTR-transfected cells, the activity of the DIDS-inhibitable Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger was studied. 8-Bromoadenosine 3',5'-cyclic monophosphate incubation resulted in Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activation in PS120 CFTR-transfected cells but had no effect on PS120 cells. Together, our results suggest that CFTR can enhance apoptosis in Chinese hamster lung fibroblasts, probably due to the modulation of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, resulting in a more efficient intracellular acidification.

Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger; DNA fragmentation; cystic fibrosis conductance regulator


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

APOPTOSIS IS A GENETICALLY DETERMINED physiological process that leads to defensive cell death, allowing the elimination of damaged or old cells from the organism. It is now well established that alterations in apoptosis contribute to the pathogenesis of several human diseases (45). Of these diseases, cystic fibrosis (CF), which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, may involve perturbation of epithelial cell apoptosis. A role of CFTR in apoptosis was first suggested based on the observation that CF patients exhibit high-molecular-weight DNA in the viscous mucus secretions of airway epithelia. The presence of this high-molecular-weight DNA suggests that it has been released in the extracellular spaces by necrotic cells that are unable to achieve DNA fragmentation and condensation. In normal secretory epithelia, programmed cell death may induce cytoplasmic acidification, which activates an acidic endonuclease allowing the cleavage, condensation, and packaging of DNA into apoptotic bodies that would be phagocytised. In contrast, in CF epithelia the impairment of this process leads to the release of large DNA fragments by senescent cells that are not phagocytised, and, therefore, enhances the viscosity of the mucus (14). This hypothesis is supported by the beneficial effect of inhaled DNase I treatment that improved the respiratory state of CF patients (37). The release of high-molecular-weight DNA fragments could be the consequence of apoptosis alteration in CF. Several authors have postulated that in CF, defective apoptosis arises from the inability of the cells to achieve the intracellular acidification necessary to activate acid endonucleases (4, 14, 26). On the other hand, different reports have described a decrease in intracellular pH (pHi) when the cells were treated with apoptosis inducers (7, 15, 24, 32). A possible hypothesis that could reconcile these findings is that an optimal apoptotic process can be linked to the magnitude of the intracellular acidification achieved during apoptosis.

It has been demonstrated that CFTR functionally interacts with the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (22, 23, 27). Therefore, it can be tempting to hypothesize that CFTR could participate in the control of apoptosis via an activation of this exchanger, leading to an improved cytoplasmic pH decrease (15). To study the potential influence of CFTR on pHi regulation during apoptosis induction, we took advantage of the PS120 Chinese hamster lung fibroblast cell line, which is devoid of the Na+/H+ exchanger (36) and does not express CFTR. This cell line also expresses a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> antiporter, and, therefore, it is very convenient to evaluate the effect of CFTR on the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> antiporter since CFTR can easily be expressed in these cells, and no compensatory effect of the Na+/H+ exchanger is present.

To examine the role of CFTR on programmed cell death, both cell lines were stably transfected with a human cDNA CFTR, and lovastatin-induced apoptosis was determined using different technical approaches. On the other hand, the NHE1 isoform of the Na+/H+ exchanger can be over expressed in PS120 cells, with or without CFTR transfection, making it possible to study how blocking cytosolic acidification affects apoptosis induction.

The results presented in this article show that controlling pH through the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> antiporter and the Na+/H+ exchanger is crucial for efficient apoptosis induction and for identification of CFTR as a key regulatory membrane protein in this process.


    MATERIALS AND METHODS
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Cell Culture and Transfection

The original CCL-39 Chinese hamster fibroblasts and the derived PS120 cell lines were grown in Dulbecco's modified Eagle's medium containing 15 mM NaHCO3, supplemented with 50 mg/ml streptomycin, 50 U/ml penicillin, and 7.5% fetal calf serum at 37°C in a humidified atmosphere of 5% CO2-95% air.

PS120 cell line. This cell line is a mutant Chinese hamster fibroblast cell line that lacks Na+/H+ exchange activity (36).

PS120 NHE1 cell line. This cell line was obtained by transfection of PS120 cells with a plasmid encoding for the NHE1 isoform of the human Na+/H+ antiporter. Transfected PS120 fibroblasts were submitted to 1-h-long 50 mM NH<UP><SUB>4</SUB><SUP>+</SUP></UP> loading (38), followed by a rapid rinse and a 1-h recovery in a medium containing 120 mM NaCl. This procedure allows the transfectants, which express a functional Na+/H+ exchanger, to survive the NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced acute acidification, whereas the nontransfected cells are killed by this procedure (35). Similar tests were repeated twice a week until clones stably expressing NHE1 were obtained.

CCL-39 CFTR, PS120 CFTR, and PS120 NHE1 CFTR cell lines. CCL-39, PS120, and PS120 NHE1 cell lines stably expressing CFTR were generated by Lipofectamine-mediated transfection with constructs containing the full-length cDNA encoding human wild-type CFTR. These constructs were obtained by transferring the 4.5-kb CFTR cDNA excised from the pTG5960 plasmid (Transgène) in the polycloning site of the eukaryote expression vector pCB6. pCB6 is a 6.2-kb vector that possesses the neomycin resistance gene and a cloning site that is under the control of the cytomegalovirus promoter. To facilitate the introduction of the insert in the MluI cloning site of the pCB6 vector, the unique restriction sites SacI and PstI flanking the coding sequence of CFTR were transformed in the MluI site. The resulting pCB6 CFTR plasmid was transfected into PS120 and PS120 NHE1 cells using Lipofectamine according to the protocol provided by the manufacturer (Life Technologies, Cergy Pontoise, France). CCL-39 CFTR, PS120 CFTR, and PS120 NHE1 CFTR transfectants were isolated by growth in media containing G418. CCL-39 mock, PS120 mock, and PS120 NHE1 mock are cell lines that were stably transfected with the empty expression vector.

Rapid Screening of the Transfectants

The G418 selection procedure enabled us to obtain nine CCL-39 clones, 11 PS120 CFTR clones, and 15 PS120 NHE1 CFTR clones. To rapidly select the clones that exhibited the best expression of CFTR, a screening procedure based on the fluorimetric measurement of membrane potential was used. Changes in membrane potential of different cells were monitored with the fluorescent dye bis(1,3-diethylthiobarbiturate)-trimethineoxonol (Bisoxonol; Molecular Probes). Fluorescence emission of this dye increases with the membrane depolarization. All the experiments were performed in a Perkin Elmer LS-5 spectrofluorimeter connected to a recorder. Buffer (1.5 ml) was added to a quartz cuvette maintained at 37°C. Bisoxonol was prepared from a stock solution (15 mM in ethanol) at a concentration of 0.15 mM in H2O and added to the cuvette to give a final concentration of 1.5 µM. For each measurement, 106 cells were added and continuously stirred with a magnetic stirrer. The fluorescence signal was recorded with excitation at 540 nm (5-nm slit width) and emission at 580 nm (10-nm slit width). Changes in fluorescence were expressed as a percentage of the control value. The clones that exhibited a significant depolarization after the addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) were considered CFTR positive and were used for further molecular and functional characterizations.

Molecular Identification of CFTR in Transfected Cell Lines

Reverse transcription and PCR amplification were performed using standard protocols in a thermal cycler (Techne). Total RNA was prepared from PS120 mock, PS120 CFTR, PS120 NHE1 mock, and PS120 NHE1 CFTR cells (2 × 106 cells) by using a micro RNA isolation kit (Tri InstaPur, Eurogentec) according to the manufacturer's directions. Primers were chosen to amplify a sequence of 297 bp localized between exon 10 and exon 12 of human CFTR. Reverse transcription was accomplished with recombinant Moloney murine leukemia virus reverse transcriptase (RT-MMLV, Life Technologies). Briefly, 15 µg of RNA were dissolved in 6 µl of water and mixed with 0.25 µM oligo 5'-CCATGAGTTTTGAGCTAAAGTCTGGC-3' (oligo A). The mixture was heated for 2 min at 70°C, chilled on ice, and completed to a volume of 20 µl to obtain a final reaction mixture containing 50 mM Tris · HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 0.5 mM dNTP, and 10 mM dithiothreitol. The solution was incubated for 90 min at 37°C after addition of 400 units of reverse transcriptase. Five microliters of the reaction was then added to a 100-µl volume of the PCR mixture containing 20 mM Tris · HCl, pH 8.4, 50 mM KCl, 1 mM MgCl2, 0.025 µM oligo 5'-GTTCTTGGAGAAGGTGGAATCACA-3' (oligo B), and 0.025 µM oligo A (see above). The mixture was incubated for 4 min at 94°C, and 5 units of Taq polymerase (Life Technologies) was added. The conditions for amplification were as follows: each cycle consisted of incubation at 94°C for 2 s, 60°C for 2 s, and 72°C for 10 s, for a total of 30 cycles. At the end of this series, the reaction was incubated at 72°C for 10 min. Controls were performed without RT-MMLV and also without RNA. All buffers were prepared in diethyl pyrocarbonate-treated water. After RT-PCR, 10-20 µl of each reaction mixture was subjected to electrophoresis on a 0.8% agarose gel to size fractionate the RT-PCR products.

The PCR-amplified fragments were subsequently cloned in the pGEM vector using Promega pGEM-T easy cloning kit. Plasmid DNA containing the 297-bp insert was then sequenced using oligonucleotides A and B (see above) as sequencing primers.

Functional Identification of CFTR in Transfected Cell Lines

Intracellular Cl- measurements. cAMP-dependent Cl- fluxes were assessed using the halide-sensitive fluorescent probe 6-methoxy-N-ethylquinolinium chloride (MEQ) according to the protocol developed by Biwersi and Verkman (6). Transfected cells (24-h-old) grown in petri dishes were loaded for 10 min at 37°C with 5 mM 6-methoxy-N-ethyl-1,2-dihydroquinoline (diH-MEQ) added to the culture medium. Dishes were carefully rinsed with NaCl solution containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, 5 glucose, and 20 HEPES, pH 7.4, and were then placed on an inverted microscope stage. Quantitative measurements of MEQ fluorescence were made with an optical system composed of a Zeiss ICM-405 inverted microscope with a Zeiss 40 objective (achromat oil 40/0.85). Fluorescence excitation was provided by a 75-W xenon lamp (Osram) and was regulated by a computer-controlled shutter (Uniblitz). The excitation beam was filtered through narrow band filters (350 nm; Oriel) mounted in a motorized wheel (Lambda 10-2; Sutter Instrument) equipped with a shutter to control the exposure times. The incident and the emitted fluorescence radiation beams were separated through a Zeiss chromatic beam splitter. Fluorescence emission was selected through a 490-nm narrow band filter (Oriel). The transmitted light images were viewed by an intensified camera (Extended-ISIS; Photonic Science, Sussex, UK). The eight-bit Extended-ISIS camera was equipped with an integration module to maximize the signal-to-noise ratio. The video signal from the camera proceeded to an image processor integrated in a DT-2867 image card (Data Translation) installed in a Pentium 100 PC. The processor converts the video signal into 512 lines by 768 square pixels/line by eight bits per pixel. The eight-bit information for each pixel represents one of the 256 possible gray levels, ranging from 0 (for black) to 255 (for white). Image acquisition and analysis were performed by the 2.1 version of AIW software (Axon Instruments). The final calculations were made using Excel software (Microsoft).

Relative rates of influx and efflux were computed from the time course of intracellular fluorescence and were expressed as relative fluorescence variations using
(&mgr;F&cjs0823;  d<IT>t</IT>)&cjs0823;  F<SUB>0</SUB><IT> · </IT>min<SUP>−1</SUP>
where µF/dt is the initial rate of fluorescence change upon addition or removal of Cl-, and F0 is MEQ fluorescence in the presence of 140 mM potassium thiocyanate (KSCN).

Cl- efflux was induced by isosmotic replacement of the NaCl solution by NaNO3 solution containing (in mM) 140 NaNO3, 5 KNO3, 3 calcium gluconate, 1 MgSO4, 5 glucose, and 20 HEPES, pH 7.4. To determine the fluorescent background, at the end of each experiment, PS120 transfected cells were incubated in 140 mM KSCN, which rapidly quenched the MEQ fluorescence.

Whole cell experiments. Whole cell currents were recorded from 24-h-old PS120 transfected cells grown in petri dishes maintained at 35°C for the duration of the experiments. The ruptured-patch whole cell configuration of the patch-clamp technique was used. Patch pipettes (resistance 2-3 MOmega ) were made from borosilicate capillary tubes (1.5-mm outer diameter, 1.1-mm inner diameter; Clay Adams) using a two-stage vertical puller (PP 83; Narishige, Tokyo, Japan) and filled with a solution containing (in mM) 140 N-methyl-D-glucamine (NMDG) Cl-, 5 EGTA, 5 ATP, and 10 HEPES, pH 7.4. The bath solution contained in (mM) 140 NaCl, 1 CaCl2, 60 mannitol, and 10 HEPES, pH 7.4. Cells were observed by using an inverted microscope, the stage of which was equipped with a water robot micromanipulator (WR 89, Narishige). The patch pipette was connected via an Ag-AgCl wire to the headstage of an RK-400 patch amplifier (Biologic). After formation of a gigaseal, the fast compensation system of the amplifier was used to compensate for the headstage intrinsic input capacitance and the pipette capacitance. The membrane was ruptured by additional suction to achieve the conventional whole cell configuration. At this stage, the cell capacitance was compensated for by using settings available on the RK-400 amplifier. No series resistance compensation was applied, but experiments in which the series resistance was higher than 20 MOmega were discarded. Solutions were perfused in the extracellular bath by using a four-channel glass pipette, the tip of which was placed as close as possible to the clamped cell.

Voltage-clamp commands, data acquisition, and data analysis were controlled by a computer equipped with a Digidata 1200 interface (Axon Instruments). pCLAMP software (versions 5.51 and 6.0, Axon Instruments) was used to generate whole cell current-voltage (I-V) relationships, with the membrane currents resulting from voltage stimuli being filtered at 1 kHz, sampled at 2.5 kHz, and stored directly on hard disk. Cells were held at a holding potential (V hold) of -50 mV, and 400-ms pulses from -100 to +120 mV were applied with increments of 20 mV every 2 s.

In Situ Apoptosis Evaluation

Apoptosis induction. Lovastatin-induced apoptosis was studied in CCL-39 mock, CCL-39 CFTR, PS120 mock, PS120 CFTR, PS120 NHE1 mock, and PS120 NHE1 CFTR cell lines. Lovastatin was dissolved in DMSO and kept in a stock solution of 4 mg/ml. The quantity of DMSO added to the incubation solutions never exceeded 0.01%. For kinetics studies, cell lines grown in petri dishes were incubated for 12, 16, 20, 30, or 40 h with 10 µM lovastatin added in the culture medium containing 15 mM NaHCO3. Parallel control experiments were performed by incubating the cells with 0.01% DMSO only instead of lovastatin. In another series of experiments, the external pH of the culture medium was increased by adding 20 mM HEPES buffered at pH 8.0, and the cells were incubated in this medium for 20, 30, and 40 h with 10 µM lovastatin.

Morphological counting of apoptotic cells. CCL-39 and PS120 transfected cells were grown in 35-mm petri dishes as described above. After the appropriate time of incubation with the apoptosis inductor (lovastatin), living cells were carefully washed with fresh culture medium and incubated 10 min in the presence of 100 µM Hoechst-33258 and propidium iodide. Nuclei were visualized with a fluorescence microscope using excitation 348 nm/emission 480 nm wavelength for Hoechst-33258 and excitation 500 nm/emission 640 nm wavelength for propidium iodide. Micrographs (color slides) were taken at each wavelength. Afterward, the preparation was washed and stained with 10 µl of orcein solution (1 g orcein, 10 ml 70% ethanol, 600 µl, and 12 N HCl). Micrographs of orcein-stained cells were then taken. Thus in a given culture, the same zone was visualized after staining with Hoechst-33258 and propidium iodide and after staining with orcein. Apoptotic cells were counted by comparing the three stainings. A cell was considered apoptotic only if the nuclei were not stained by propidium iodide and presented chromatin condensation with visible apoptotic bodies. The counts of apoptotic nuclei were performed directly on the micrographs. Between 100 and 200 cells were scored by four different observers who were blinded to the culture conditions. The number of cells with DNA condensation was expressed as the percentage of total cells.

Annexin V labeling. Apoptosis was also detected using the Apoalert annexin V apoptosis kit (Clontech kit K2025). For this purpose, PS120 cells were grown in 35-mm petri dishes. After lovastatin treatment, plated cells were rinsed with the binding buffer and incubated for 15 min in the dark at room temperature with annexin V-FITC conjugate (final concentration: 0.8 µg/ml) and propidium iodide (final concentration: 0.5 µg/ml). The cells were observed under a fluorescence microscope using a dual filter set for FITC and rhodamine (Olympus BH T2). Cells that had bound annexin V showed green staining in the plasma membrane. Cells that had lost membrane integrity exhibited red staining throughout the cytoplasm and diffuse green staining on the cell surface. Red- and green-stained cells were counted on micrographs. The percentage of apoptotic cells was calculated by comparing the number of pure green-stained cells to the total number of cells counted on phase-contrast micrographs. The counts were performed in a blind manner as described above.

Quantitative Analysis of Fragmented DNA

DNA fragmentation was assayed as previously reported (11, 18). After lovastatin treatment, cells grown in petri dishes were lysed by the addition of 500 µl of cold lysis buffer (7.5 mM Tris · HCl, 1.5 mM EDTA, and 0.25% Triton X-100, pH 8.0) for 30 min at 4°C. The lysates were centrifuged at 13,000 g for 30 min at 4°C to separate intact chromatin (pellet) from DNA fragments (supernatants). Pellets were resuspended in 500 µl of TE buffer (10 µM Tris · HCl and 1 mM EDTA, pH 7.5), and the DNA contents of pellets and supernatants were measured by a modification of the fluorimetric micromethod of Switzer and Summer (44) or by the PicoGreen DNA quantitation reagent (Molecular Probes kit P-7581). The percentage of DNA fragmentation was calculated by the ratio of the fragmented DNA in the supernatant over the total DNA.

Analysis of DNA Fragmentation in Agarose Gel

PS120 transfected cells were grown in 100-mm petri dishes and treated with lovastatin (40 µg/ml) for 40 h. At the end of the treatment, cells were lysed at 4°C for 30 min in buffer containing 10 mM Tris-EDTA and 0.2% Triton X-100, pH 8.0. After RNase treatment (100 µg/ml) for 30 min at 37°C, the lysate was incubated in proteinase K (100 µg/ml) for 30 min at 37°C. DNA was precipitated with 0.5 M NaCl in isopropanol overnight at -20°C. The precipitate was centrifuged at 13,000 g for 10 min, washed with 70% ethanol solution, and allowed to dry. DNA was dissolved in 50 µl of Tris-EDTA buffer. DNA samples were loaded onto 1% agarose gels and run at 100 V for 1 h.

pHi Measurements

The fluorescent pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure cytosolic pH, as described in detail previously (5). Quantitative digital measurements were made as described earlier.

PS120 transfected cells grown in 35-mm petri dishes were incubated with 4 µM BCECF-AM at 37°C for 15 min in a humidified atmosphere of 5% CO2-95% air. Loaded cells were carefully rinsed and placed on the stage of an inverted microscope. The cells were excited successively at 490 and 450 nm, and each pair of images was digitized and stored on the hard disk of a computer. The basal pHi was determined on 10 images recovered every 20 s in the control solution. Cells were then perfused with the different test solutions, and images were successively recorded every 20 s. At the end of each experiment, the fluorescence signals relating to pHi changes were calibrated using the K+/H+ exchange ionophore nigericin. For this purpose, the cells were perfused with KCl solutions (in mM) that comprised 140 KCl, 1 CaCl2, 20 HEPES, and 10 µM nigericin, the pH of which was adjusted to 8.0, 7.5, 7.0, and 6.5, respectively, with Tris. The initial rate of change in pHi (Delta pHi/min) was measured with Microsoft Excel software, using linear regression analysis on the traces. To ensure an adequate renewal of the medium, the solutions were perfused at a rate of 2 ml/min.

To determine the activity of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger at physiological pH values, cells were first incubated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered NaCl solution containing (in mM) 125 NaCl, 15 NaHCO3, 5 KCl, 1 CaCl2, 5 glucose, and 20 HEPES, pH 7.4. The solution was then replaced by a Cl--free solution containing (in mM) 125 sodium gluconate, 15 NaHCO3, 5 potassium gluconate, 3 calcium gluconate, 5 glucose, and 20 HEPES, pH 7.4. Under these conditions, Cl- leaves the cell in exchange for extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and the cytosolic pH increases.

Chemical Compounds

Cell culture media and nutrients were obtained from GIBCO BRL (Basel, Switzerland). Lovastatin and Hoechst-33258 were obtained from Calbiochem (France Biochem, Meudon, France). Orcein, forskolin, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), DIDS, and 8-Br-cAMP were obtained from Sigma Aldrich (Saint Quentin Fallavier, France). Apoalert annexin V was obtained from Clontech Ozyme (Montigny le Bretonneux, France). PicoGreen and BCECF were obtained from Molecular Probes (Leiden, Netherlands). Cariporide (HOE-642) was a gift of Adventis (France).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Biochemical and Functional Characterization of PS120 Cells Expressing CFTR

With the use of pCB6-CFTR constructs, several antibiotic-resistant clones of PS120 and PS120 NHE1 cells were obtained. To select the clones that expressed CFTR, we used the oxonol technique to measure the relative changes of membrane potential induced by cAMP. Thus we selected one PS120 and one PS120 NHE1 clone that exhibited a stronger depolarization after 8-Br-cAMP application. These clones were named PS120 CFTR and PS120 NHE1 CFTR and were analyzed further for CFTR biochemical and functional expression.

PS120 CFTR and PS120 NHE1 CFTR total RNA was reverse transcribed and amplified by PCR using A and B primers. These primers amplify a 297-bp stretch of sequence situated between exon 10 and exon 12 of the human CFTR gene. An analysis of the RT-PCR products by electrophoresis on agarose gels stained with ethidium bromide revealed only one product of ~300 bp (Fig. 1) in RNA extracts. An identical analysis without prior reverse transcription of the RNA sample revealed no amplification of any product. The PCR products obtained from transfected cells were sequenced and found to share 100% identity with the appropriate region on the human CFTR mRNA. This indicated that the CFTR gene was stably transfected and transcribed in these clones.


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Fig. 1.   Reverse transcriptase-polymerase chain reaction (RT-PCR) products using primers specific for human cystic fibrosis transmembrane conductance regulator (CFTR) generated from cDNA from cultures of PS120 cells. Primers between exon 10 and exon 12 (primers A and B) generated a band of 300 bp both in PS120 CFTR (lane 1) and PS120 Na+/H+ exchanger (NHE1) CFTR (lane 3). No amplification was observed both in PS120 mock (lane 2) and PS120 NHE1 mock (lane 4) cells. Molecular weight markers (fX 174 + HaeIII) were run in parallel at the right edge of the agarose gel (lane 0).

To confirm CFTR functional expression, we used a cell membrane halide-permeability assay with the Cl- indicator fluorescent dye diH-MEQ. The Cl- permeabilities of cell membranes were estimated by measuring intracellular MEQ fluorescence using video microscopy. The flow of Cl- across the membrane was assessed by the addition or removal of Cl- from the bathing solutions. Initial relative Cl- efflux and influx rates are given in Fig. 2. The application of 8-Br-cAMP (1 mM) significantly increased the initial Cl- efflux and influx only in CFTR-transfected PS120 NHE1 and PS120 cells. In contrast, the nucleotide did not modify Cl- fluxes in PS120 mock-transfected cells. Hence, expression of CFTR in PS120 cells induced cAMP-activated Cl- permeability.


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Fig. 2.   Effects of 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) on relative Cl- efflux and influx through PS120 cells loaded with 6-methoxy-N-ethyl-1,2-dihydroquinoline. Cl- efflux was induced by replacement of NaCl solution with NaNO3 solution, and Cl- influx was induced by removal of NaNO3 solution. Solid bars: Cl- flux values in control conditions; hatched bars: Cl- flux values in the presence of 8-Br-cAMP (1 mM). Values are means ± SE of n cell cultures. NS, not significantly different from control values. ***P < 0.0001 significantly different from control values (paired t-test).

To ensure that this increase in Cl- fluxes was due to the activation of Cl- conductance, whole cell clamp experiments were also performed. Whole cell currents were recorded with Ca2+-free pipette solutions containing 140 mM NMDG Cl-, whereas hyperosmotic extracellular solutions contained 140 mM NaCl and 50 mM mannitol. In PS120 mock- and PS120 CFTR-transfected cells, the voltage step protocol elicited small currents (Fig. 3A) that changed linearly with the membrane voltage. By contrast, only PS120 CFTR cells exposed to 1 mM 8-Br-cAMP exhibited an increase in membrane currents (Fig. 3B), with the maximum increase obtained 3-4 min after the onset of perfusion. Figure 3E shows that the activated currents presented a linear I-V relationship that reversed close to 0 mV. Under these conditions, the current amplitude at +100 mV reached 463 ± 53 pA, and the mean conductance was 5.1 ± 0.6 nS (n = 3 cell cultures).


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Fig. 3.   Characteristics of 8-Br-cAMP-induced whole cell Cl- currents in PS120 CFTR-transfected cells. With a hyperosmotic NaCl solution in bath and a N-methyl-D-glucamine Cl- solution in pipette, membrane potential was held at -50 mV and stepped to test potential values between -100 and +120 mV in 20-mV increments. Whole cell currents were measured from unstimulated cells (A), in the presence of a bath solution consisting of 1 mM 8-Br-cAMP alone (B), in the presence of 8-Br-cAMP with I- (C), after removal of I- (D), or in the presence of 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; E). F: average current-voltage relationships measured 390 ms after onset of pulse, obtained from same cell at rest, during 8-Br-cAMP stimulation alone and after Cl- substitution by I-. Values are means ± SE of 11 cells obtained from 3 cell cultures.

The experiments yielding these data were performed in symmetrical Cl- concentrations in the presence of EGTA in the pipette to avoid involvement of intracellular Ca2+ and in hyperosmotic bath solutions to block swelling-activated currents. The reversal potential was very close to that of Cl-, and, in the absence of permeable cations in the pipette, indicated that the current was carried by Cl-.

Overall, these results strongly suggest CFTR functional expression. To confirm this, we investigated the ion selectivity of the cAMP-induced Cl- conductance. For this purpose, all except 2 mM of the Cl- in the bath solution was replaced with I-. Figure 3C shows typical recordings of the currents obtained in the presence of I-, and Fig. 3E shows I-V relations for this current carrier. Replacing external Cl- with I- strongly decreased both inward and outward currents (currents at +100 mV = 95 ± 2 pA, conductance = 1.5 ± 0.2 nS, n = 3) and shifted the reversal potential toward more positive potentials (Erev = +10.2 ± 2.0 mV). The relative permeability P<UP><SUB>I</SUB><SUP>−</SUP></UP>/P<UP><SUB>Cl</SUB><SUP>−</SUP></UP> calculated using the Erev values was 0.66 ± 0.11 (n = 3). As illustrated in the recording of Fig. 3, the effect of I- was reversible (currents at +100 mV = 300 ± 6 pA, conductance = 3.7 ± 0.3 nS, n = 3). To further characterize the Cl- current, we tested the effect of the anion channel blocker NPPB that was added to the bathing solution. As illustrated in Fig. 3E, the addition of 0.1 mM NPPB inhibited the whole cell Cl- currents within 2 min. The effect of this blocker was reversible upon washing (data not given). The I-V relationship is given in Fig. 3. Overall, 0.1 mM NPPB inhibited reversibly both inward and outward currents.

The experiments illustrated in Fig. 3 were carried out on PS120 CFTR-transfected cells. Identical results were obtained with the clone PS120 NHE1 CFTR (data not shown). The control mock-transfected cells did not present any Cl- conductance induced by cAMP, and no PCR amplification product could be detected.

Time Course of Lovastatin-Induced Apoptosis in Different Clones

Cell counting experiments. Figure 4A shows the Hoechst-33258 and propidium iodide stainings of PS120 CFTR cells before the addition of lovastatin, an efficient apoptosis inducer in fibroblasts. The nuclei excluded propidium iodide and exhibited a normal morphology with Hoechst-33258 labeling with a diffuse staining of the normal chromatin. This pattern was also observed in the other cell lines, i.e., PS120 mock-, PS120 NHE1 mock-, and PS 120 NHE1 CFTR-transfected cells. After the addition of 10 µM lovastatin for 30 h, the Hoechst-33258 staining revealed that although several nuclei still displayed a normal morphology, other cells exhibited very intense staining of condensed and fragmented chromatin (Fig. 4C). Both types of cells were not stained by propidium iodide, indicating preservation of the plasma membrane (Fig. 4C). In addition, dead cells possessing propidium iodide-labeled nuclei were also detected in this preparation (Fig. 4E). The condensation and the fragmentation of DNA clearly show that lovastatin induces programmed cell death in live PS120 fibroblasts. These characteristic properties could also be observed independently with orcein staining. As shown with Hoechst-33258 staining, control cells that were not submitted to lovastatin treatment did not exhibit chromatin condensation (Fig. 4B). By contrast, a dense and thin crown of nuclear coloration, typical of chromatin condensation, could be observed in the lovastatin-treated cells (Fig. 4D).


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Fig. 4.   PS120 CFTR-transfected cells stained with Hoechst-33258, propidium iodide, and orcein. In the absence of lovastatin, PS120 CFTR cells exhibited normal nuclear morphology revealed by Hoechst-33258 staining (A) or orcein labeling (B). After the addition of 10 µM lovastatin for 30 h, cells stained with Hoechst-33258 presented very bright staining of condensed and fragmented chromatin (open arrows in C). The same cells labeled with orcein exhibited a thin crown of nuclear coloration corresponding to chromatin condensation (solid arrows in D). These cells excluded propidium iodide (E). Some necrotic cells were stained with propidium iodide (open arrow in E).

On the basis of these morphological criteria, the time course of lovastatin-induced apoptosis was followed in the different transfected cell lines. Figure 5 shows the percentage of apoptotic cells determined after 12, 16, 20, 30, and 40 h of incubation with 10 µM lovastatin. In all cell lines, the percentage of apoptotic cells increased significantly from 12 h after the beginning of the lovastatin treatment. Interestingly, we observed that after 20 h, the percentage of apoptotic PS120 CFTR cells exhibited a much faster increase than the percentage of apoptotic PS120 mock cells. This culminated at 40 h, where we could show that this percentage was two times greater in PS120 CFTR-transfected cells than in PS120 mock cells (percentage of apoptotic cells at 40 h: PS120 mock = 23.4 ± 2.7, n = 10; PS120 CFTR = 40.4 ± 3.6, n = 11; P < 0.0001; Fig. 5A). The addition of 0.1 mM DIDS to the incubation medium strongly inhibited the effect of lovastatin in both cell lines. After 40 h of incubation with the stilbene derivative, the percentage of apoptotic cells was <10% of the cell population without significant differences between mock- and CFTR-transfected PS120 cells (Fig. 5A). A similar result was obtained when the pH of culture medium was increased. Figure 5B shows that raising the external pH to 8.0 considerably reduced lovastatin-induced apoptosis in both cell lines.


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Fig. 5.   Time course of apoptosis induced by 10 µM lovastatin added in the culture medium. In A, D, and E, apoptosis was determined with or without lovastatin (LOVA) treatment. Apoptotic nuclei were counted in 100-200 orcein-stained cells and expressed as a percentage of total cells. A: lovastatin-induced apoptosis in PS120 CFTR- and PS120 mock-transfected cells. In this series, apoptosis was also measured in the presence of 0.1 mM DIDS. B: lovastatin-induced apoptosis in PS120 CFTR- and PS120 mock-transfected cells incubated in a culture medium buffered at pH 8.0. C: lovastatin-induced apoptosis in CCL-39 CFTR- and CCL-39 mock-transfected cells. D: lovastatin-induced apoptosis in PS120 NHE1 CFTR- and PS120 NHE1 mock-transfected cells. E: lovastatin-induced apoptosis in PS120 NHE1 mock-transfected cells in the presence of 30 µM cariporide. In F, apoptosis was measured in PS120 CFTR- and PS120 mock-transfected cells after staining the cells with annexin V. Values are means ± SE of n different cell cultures.

Lovastatin-induced apoptosis was also studied in the parental CCL-39 cell line, transfected or not transfected, with CFTR (Fig. 5C). As it was observed in PS120 cells, the percentage of apoptotic cells was strongly increased in the CCL-39 cells transfected with CFTR (percentage of apoptotic cells at 40 h: CCL-39 mock = 16.6 ± 0.7, n = 12; CCL-39 CFTR = 41.0 ± 4.5, n = 12; P < 0.0001; Fig. 5C). In the cells expressing the NHE1 Na+/H+ exchanger at physiological levels, it is interesting to note that the percentage of apoptotic cells is significantly lower at 40 h in CCL-39 mock cells than in the PS120 mock cells (CCL-39 mock = 16.6 ± 0.7, n = 12; PS120 mock = 23.4 ± 2.7, n = 10; P < 0.02; Fig. 5C).

In PS120 NHE1 mock- and PS120 NHE1 CFTR-transfected cells, lovastatin enhanced apoptosis with a similar time course. However, in these cell lines, the presence of CFTR transcripts did not modify the percentage of apoptotic cells (percentage of apoptotic cells at 40 h: PS120 NHE1 mock = 19.4 ± 2.8, n = 8; PS120 NHE1 CFTR = 17.2 ± 1.3, n = 8; not significant; Fig. 5D). Finally, for each of the clones under study, the percentage of apoptotic cells remained very low in control cells not treated with lovastatin.

Because PS120 NHE1 cells overexpressed the NHE1 isoform of the Na+/H+ antiporter, we therefore performed experiments to study the influence of a potent NHE1 blocker (cariporide) on lovastatin-induced apoptosis. As illustrated in Fig. 5E, the PS120 NHE1 mock cells treated with 30 µM cariporide during lovastatin incubation exhibited a percentage of apoptotic cells significantly higher than that which was obtained in the absence of the antiporter inhibitor. In the absence of lovastatin treatment, the incubation of the cells with cariporide for 20 and 40 h only slightly increased the number of apoptotic cells (Fig. 5E).

To further study the lovastatin-induced apoptosis in PS120 cells, annexin V-labeling experiments were performed. The results reported in Fig. 5F clearly show that the percentage of cells stained by annexin V was significantly higher in PS120 CFTR- than in PS120 mock-transfected cells, both after 30 or 40 h of incubation with 10 µM lovastatin (percentage of annexin V-positive cells at 40 h: PS120 mock = 18.2 ± 1.8, n = 7; PS120 CFTR = 36.2 ± 2.7, n = 7; P < 0.0001; Fig. 5F).

DNA fragmentation measurements. To further demonstrate lovastatin induced-apoptosis in PS120 transfected cells, fragmentation of DNA was studied. Figure 6 illustrates the DNA fragmentation patterns on an ethidium bromide-stained agarose gel produced 40 h after exposure of PS120 CFTR cells to 10 µM lovastatin. The characteristic ladder of DNA fragmentation, indicative of internucleosomal DNA cleavage, was observed only in cells incubated with lovastatin.


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Fig. 6.   Agarose gel of DNA fragmentation. PS120 mock- and PS120 CFTR-transfected cells were exposed to 10 µM lovastatin for 40 h. Control experiments without lovastatin treatment were performed using PS120 CFTR cells. DNA size markers are shown (right).

To quantify DNA fragmentation, each clone was treated with 10 µM lovastatin and harvested at the indicated times. DNA was then assayed by fluorimetric methods. Compared with control conditions, a significant amount of DNA fragmentation was detected after 16 h of lovastatin incubation. From this time onward in all cell lines, DNA fragmentation increased markedly with the incubation times. However, examination of the PS120 cells (Fig. 7A) clearly indicates that CFTR transfection resulted in a stronger fragmentation at every incubation time. By contrast, this is not the case in PS120 NHE1 cells in which the percentage of fragmented DNA was identical to mock- or CFTR-transfected cells (Fig. 7B).


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Fig. 7.   Time course of DNA fragmentation induced by 10 µM lovastatin in PS120 CFTR- and PS120 mock-transfected cells (A) and in PS120 NHE1 CFTR- and PS120 NHE1 mock-transfected cells (B). In each cell line, DNA fragmentation was also determined in the absence of lovastatin treatment. Values are means ± SE of n different cell cultures.

pHi During Lovastatin-Induced Apoptosis

To determine the key proteins implicated in the control of the pH decrease during apoptosis, we then monitored the pH changes in our various cell lines after addition of lovastatin. The results are illustrated in Fig. 8, A and B. In the absence of lovastatin, pHi did not differ between one cell line to another and did not significantly vary with time (0 min: pHi = 7.38 ± 0.10; 40 h: pHi = 7.37 ± 0.09; n = 7; Fig. 8). After 20 h of incubation with 10 µM lovastatin, a decrease of ~0.25 pH units was observed in all cell lines. After 40 h of incubation, the pHi of PS120 mock, PS120 NHE1 mock, and PS120 NHE1 CFTR was not significantly different from that measured at 20 h. By contrast, treatment of PS120 CFTR cells with lovastatin for 40 h caused the pHi to strongly drop below the pHi value determined at 20 h (20 h: pHi = 7.30 ± 0.04; 40 h: pHi = 6.85 ± 0.10; n = 5, P < 0.001). As illustrated in Fig. 8A, this significant decrease of pHi was completely impaired in PS120 CFTR cells incubated concomitantly with 10 µM lovastatin and 0.1 mM DIDS (20 h: pHi = 7.18 ± 0.07; 40 h: pHi = 7.24 ± 0.09; n = 10, not significant). Moreover, increasing the pH of the culture medium to a value of 8.0 also prevented the decrease of cytoplasmic pH during lovastatin treatment in both PS120 mock- and PS120 CFTR-transfected cells (Fig. 8A).


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Fig. 8.   Effect of lovastatin on the cytoplasmic pH (pHi) of different PS120 cell lines (A and B). Cells were incubated for 20 and 40 h with 10 µM lovastatin. Fifteen minutes before the end of the incubation, cells were loaded with 2',7'-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein (BCECF), and the pH was measured using fluorescence video microscopy. Values are means ± SE of n different cell cultures.

In another experimental series, the pHi of PS120 NHE1 mock and PS120 NHE1 CFTR cells was also measured during lovastatin treatment but in the presence of cariporide (30 µM), a potent blocker of the NHE1 isoform of the Na+/H+ antiporter (39). Under these conditions, control PS120 NHE1 mock and PS120 NHE1 CFTR cells maintained a pHi of 7.19 ± 0.10 and 7.33 ± 0.02 (n = 3), respectively. These values were significantly lower than those determined in the absence of cariporide (pHi = 7.54 ± 0.07, n = 5). Incubation of the cells with lovastatin for 20 h induced a significant pHi decrease (pHi of PS120 NHE1 mock = 6.90 ± 0.04; pHi of PS120 NHE1 CFTR = 6.91 ± 0.05; n = 3). At 40 h, an additional drop of pHi was observed in both cell lines (pHi of PS120 NHE1 mock = 6.72 ± 0.06; pHi of PS120 NHE1 CFTR = 6.74 ± 0.02; n = 3; Fig. 8B).

Figure 9 shows the relationship between pHi and the percentage of apoptotic cells 40 h after lovastatin addition. As observed, there is a significant correlation between intracellular acidification and apoptosis (correlation data: y = 56 × -427, r = 0.95, P < 0.001).


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Fig. 9.   Correlation between percentage of apoptotic cells as a function of change in pHi. Cells [Na+/H+ antiporter-deficient CCL-39 cells (PS120), transfected or not transfected, with CFTR and/or overexpressing the Na+/H+ antiporter] were incubated for 40 h with 10 µM lovastatin. Fifteen minutes before the end of the incubation, cells were loaded with BCECF, and the pH was measured using fluorescence video microscopy. Apoptotic cells were counted after orcein staining.

Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Exchanger Activity in PS120 Cells

In the above experiments, the acidification of PS120 CFTR cells induced by lovastatin was clearly blocked by 0.1 mM DIDS. This observation suggested the involvement of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in the acidification phenomenon. Further experiments were therefore performed to identify such an exchanger in PS120 cells. For this purpose, PS120 mock and PS120 CFTR cells loaded with BCECF were maintained in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solutions, and the pHi was recorded by fluorescence microscopy. In the experiment illustrated in Fig. 10A, Cl- was replaced by gluconate, causing the pHi to increase over a 6-min period. This effect was reversed when the gluconate was rinsed away with a NaCl solution.


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Fig. 10.   Effects of 8-Br-cAMP on pHi regulation of PS120 cells after extracellular Cl- removal. A: time course of pHi variations. PS120 mock- and PS120 CFTR-transfected cells were loaded with BCECF and successively perfused with a Cl--free HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered solution containing sodium gluconate (Na Gluc.). The sequence of buffer substitution is indicated (top). 8-Br-cAMP (1 mM) was added to bathing medium 2-3 min before the second NaCl substitution in the presence or the absence of 0.1 mM DIDS. B: effects of 0.1 mM glibenclamide, 0.1 mM DIDS, and 0.1 mM NPPB on the rate of intracellular alkalinization induced by Cl- removal in both cell lines in the presence of 8-Br-cAMP. Values are means ± SE of n different cell cultures. ***P < 0.0001, significantly different from control values (Student's t-test).

In the second part of the experiment, the effect of removal and addition of Cl- to the incubation medium was studied in the presence of 8-Br-cAMP (1 mM). Under these conditions, the initial rate of pHi recovery was 2.3-fold more rapid in PS120 CFTR- than in PS120 mock-transfected cells (Fig. 10, A and B). This increase of pHi recovery induced by cAMP in PS120 CFTR cells was not observed when the cells were also incubated with 0.1 mM glibenclamide, and it was not modified in the presence of 0.1 mM NPPB (Fig. 10B). On the other hand, the application of 0.1 mM DIDS completely inhibited the intracellular alkalinization induced by Cl- removal in both cell lines (Fig. 10, A and B). To further examine the nature of this Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, we determined the effect of Na+ on its activity. In the absence of external Na+, the effect of removal and addition of Cl- was identical to that observed in the presence of Na+. Moreover, removal of Na+ did not impair the cAMP-induced alkalinization in PS120 CFTR cells (Delta pH/min: control = 0.40 ± 0.03; without Na+ = 0.38 ± 0.03; n = 3).

Role of Different Drugs on Lovastatin-Induced Apoptosis in PS120 Cells

The above results strongly suggest a role of CFTR in controlling apoptosis in lovastatin-treated PS120 cells. To further investigate this role, the actions of several modulators of Cl- permeability were studied. These products were tested in combination with lovastatin, and the percentage of apoptotic cells was determined after 40 h of treatment. The histograms of Fig. 11 compare the percentage of apoptotic cells in PS120 mock-transfected or PS120 CFTR-transfected cells with or without lovastatin treatment. We first investigated the effect of DIDS (0.1 mM) on lovastatin-induced apoptosis. As illustrated in Figs. 5A and 11, this stilbene derivative was a very efficient inhibitor of apoptosis in both PS120 mock- and PS120 CFTR-transfected cell lines. Together, our results imply that CFTR is involved in the control of apoptosis, possibly through its action on the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> antiporter. To determine whether the activation of this antiporter requires the Cl- conductive function of CFTR, we then studied the effect of the Cl- channel blocker NPPB. Figure 11 clearly shows that NPPB (0.1 mM) did not decrease the apoptosis levels in both cell lines. We also studied the effect of a sulfonylurea derivative, glibenclamide, which has been shown to block CFTR. In our experiments, glibenclamide (0.1 mM) application strongly reduced the lovastatin-induced apoptosis in PS120 CFTR-transfected cells only (Fig. 11).


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Fig. 11.   Effects of 0.1 mM DIDS, 0.1 mM NPPB, 0.1 mM glibenclamide (GLIB.), and 1 mM 8-Br-cAMP on apoptosis induced by 10 mM lovastatin in PS120 mock- and PS120 CFTR-transfected cells. The drugs were added together with lovastatin, and the percentage of apoptotic cells was determined by using propidium iodide and orcein labeling. Values are means ± SE of n different cell cultures. ***P < 0.0001, significantly different from control values (paired t-test).

In the last series of experiments, we examined the effect of a cAMP-elevating agent. Incubation of the cells with 8-Br-cAMP (1 mM) increased lovastatin-induced apoptosis in PS120 mock- but not in PS120 CFTR-transfected cells (Fig. 11).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this work was to investigate the putative role of CFTR in the control of apoptosis via pHi regulatory mechanisms. For this purpose, PS120 cells devoid of Na+/H+ antiporter, CCL-39 cells expressing physiological levels of NHE1, and PS120 NHE1 cells overexpressing the NHE1 isoform of the Na+/H+ antiporter were stably transfected with a cDNA encoding for the human CFTR. After selection, the clones were characterized to ensure that they expressed a normal CFTR-mediated Cl- conductance.

To induce apoptosis, we chose lovastatin. The mechanism by which lovastatin induces apoptosis is related to the inhibition of the 3-hydroxy-3-methylglutaryl coenzyme A reductase. This enzyme is the rate-limiting enzyme of cholesterol biosynthesis. It generates mevalonate, resulting in the synthesis of the noncholesterol metabolites required for cell survival. Thus the depletion of these metabolites induced by lovastatin leads to programmed cell death. Lovastatin has been shown to be an efficient apoptosis inducer in different cell types such as HL-60 leukemic cells (32) and C6 glial cells (8). However, one potential problem with this drug is that it might decrease CFTR function. In fact, Shen et al. (42) have suggested that 50 µM lovastatin could reduce the number of cAMP-dependent Cl- channels in the apical membrane of epithelial cells. To circumvent this potential problem, all experiments were carried out with a fivefold lower lovastatin concentration (10 µM), which was sufficient to induce apoptosis with a high efficiency. In addition, we verified that there was no significant difference between lovastatin-treated and lovastatin-untreated cells when measuring cAMP-induced Cl- currents. Considering that cAMP Cl- permeability reflects CFTR function, it could be reasonably concluded that under our experimental conditions, lovastatin did not interfere significantly with CFTR activity.

As expected, lovastatin treatment in all cell lines induced apoptosis in a time-dependent manner. To ensure a proper detection of the apoptotic process, we based our analysis on different criteria including nuclear staining, annexin V labeling, and DNA fragmentation. To optically separate apoptotic and necrotic cells, we used double staining with Hoechst-33258 and propidium iodide. Apoptotic cells excluded propidium iodide and presented visible condensed nuclei that appeared brightly stained by Hoechst-33258. Further labeling of these cells with orcein confirmed chromatin margination with the presence of apoptotic bodies (2). Annexin V-FITC binding was also used to detect apoptosis. Positive labeling correlates with the appearance of nuclear fragmentation and reflects a later phase of apoptosis (13). The use of all these techniques led to the observation that the CCL-39 and PS120 cells expressing CFTR underwent more apoptosis after lovastatin treatment than the cells not expressing CFTR. The number of apoptotic cells began to diverge significantly only after 20 h, being approximately twofold higher in CFTR-expressing cells after 40 h. The DNA fragmentation was also greater in CFTR-transfected cells, but this phenomenon occurred earlier and preceded the appearance of the first apoptosis images. This last observation is in accordance with published data that indicate that DNA fragmentation occurs before the formation of apoptotic bodies (10).

The participation of CFTR in the control of apoptosis has already been suggested by several works but remains controversial. Contradictory results have been reported with some studies indicating that CFTR could increase apoptosis, whereas other works suggest a reverse effect. Interestingly, the study of phenotypic abnormalities in CF epithelia has indicated the presence of inappropriately high-molecular-weight DNA fragments in lung mucous secretions, suggesting inefficient apoptosis (26). Moreover, in C127 mammary epithelial cells expressing the Delta F508 CFTR, cycloheximide-induced apoptosis was clearly decreased (15). On the contrary, a recent study performed in Hep G2 human cells indicates that CFTR inhibition induced apoptosis (21).

The present study reveals more direct evidence that CFTR increases lovastatin-induced apoptosis in our cell systems and provides us with a potential mechanism for this effect. The examination of pHi revealed that the cell lines underwent intracellular acidification during lovastatin treatment. Moreover, there is a highly significant correlation between intracellular acidification and the percentage of apoptotic cells. The most significant observation is that this acidification was stronger in PS120 CFTR cells than in all other cell lines. According to previously published data and our results, it is very likely that the increase in apoptosis levels can be correlated with this drop of pHi (3, 14, 20, 24). In fact, this acidification is probably essential to allow the cells to enter into the apoptotic cycle, and it is possible that any means of preventing cytosolic acidification strongly decreases lovastatin-induced apoptosis. To challenge this hypothesis, we decided to block this acidification by using different experimental approaches. First, we increased the pH of the culture medium, and second, we overexpressed an extremely potent acid extruder, the Na+/H+ exchanger (NHE1 isoform). Under both experimental conditions, lovastatin slightly induced apoptosis in PS120 cells, but interestingly, CFTR was unable to increase the number of apoptotic cells. Thus clamping the pH by overexpressing the Na+/H+ exchanger or increasing external pH totally abolishes this acidification and impairs the ability of CFTR to enhance lovastatin-induced apoptosis. Moreover, in PS120 cells overexpressing NHE1, the ability of cariporide to enhance lovastatin-induced apoptosis and to concomitantly decrease pHi is consistent with this hypothesis. As a corollary, it has been demonstrated that an activation of the Na+/H+ antiporter activity suppressed lovastatin-induced apoptosis in HL-60 cells (32).

To have a physiological relevance, the mechanism that we propose needs to be functional in the presence of normal levels of NHE1. Therefore, we decided to study the effect of CFTR on apoptosis in a cell line expressing physiological levels of the Na+/H+ antiporter: the CCL-39 Chinese hamster fibroblasts. In these cells, lovastatin induced apoptosis, as it was also observed in the PS120 cell lines. When compared with the PS120 mock-transfected cells, the CCL-39 mock cells exhibited a significantly lower percentage of apoptotic cells, especially at 30 and 40 h. This result suggests that in the absence of CFTR, the basal level of NHE1 can limit the apoptosis induction. By contrast, the expression of CFTR in CCL-39 cells greatly enhanced the time course of apoptosis. This result confirms our finding that CFTR can positively regulate apoptosis in a physiologically relevant cell system and suggests a negative control of CFTR on NHE1 during apoptosis induction. We propose that the expression of CFTR is sufficient to modulate NHE1 when the protein is present in physiological quantities (CCL-39 CFTR cell lines) and that there are insufficient CFTR protein molecules to interact with NHE1 when NHE1 is overexpressed at the plasma membrane (PS120 NHE1 CFTR cell line). This would render CFTR inefficient at decreasing pHi in the cells overexpressing NHE1, resulting in a defective apoptosis process for the PS120 NHE1 CFTR cells.

Of the mechanisms that participate in the regulation of pHi, the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger might be a good candidate for CFTR-induced acidification. In the PS120 cell line, the strong increase in pHi induced by external Cl- removal in an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> medium could well be mediated by the activation of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger because DIDS completely prevented this pHi recovery. Addition of 8-Br-cAMP increased the rate of alkalinization in PS120 cells expressing CFTR but not in PS120 mock cells. In the literature, several studies clearly indicate that expression of CFTR is required for regulation of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger by cAMP (22, 23, 27). However, other works show that the increase of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion generated by cAMP is mainly due to an increase of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conductance through the CFTR channel (16, 19, 33, 34). Finally, a recent study reconciled these observations by demonstrating that CFTR could function both as an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conductor and as a facilitator of membrane Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (9). In the present study, the inhibition by DIDS of the cAMP-dependent alkalinization indicates that the stimulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> fluxes did not directly occur via an anion-conductive pathway because the CFTR Cl- channels are quite insensitive to stilbene derivatives (17); therefore, the acidification is mediated by the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. In any case, the experiments performed in our system do not prove that the mechanism by which CFTR activates the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger during apoptosis involves an elevation of the cAMP levels. These experiments show only that the anion exchanger exhibits an enhanced sensitivity to second messengers in the presence of CFTR. The mechanism of the stimulation of the anion exchanger during apoptosis will most likely be the subject of future studies. It is interesting to note that the Cl- channel blocker NPPB does not modify the cAMP-sensitive acidification, showing that the action of CFTR on the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger does not involve the Cl- translocation through the CFTR channel. By contrast, the CFTR Cl- channel blocker glibenclamide completely prevented cAMP-dependent alkalinization. In the absence of external Cl-, this action could not be the direct consequence of a decrease of Cl--conductive efflux because such a diminution would increase the outward-directed Cl- gradient, allowing a better HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> entry into the cells through the exchanger. Thus glibenclamide appears to block the cAMP-induced alkalinization independently of its inhibition of the channel conductance. This raises the problem of the respective effects of both inhibitors on CFTR functions. Many studies have now demonstrated that CFTR appears to function not only as a Cl- channel but also as a channel regulator (41) and that blocking the Cl- channel activity does not necessary impair the regulatory role of the protein. It is possible that glibenclamide also blocks the regulatory function of CFTR. Such a blockade could result in the loss of the ability of CFTR to regulate the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. To reinforce this point, it is important to note that the activation of CFTR by cAMP is necessary for regulating different transporters such as the epithelial Na+ channel (43), the renal outer medulla K+ channel (30), or the KCNE3-dependent KvLQT1 K+ channel (40). In all these cases, the Cl- channel activity was not necessarily involved.

The present data clearly indicate that the increase of lovastatin-induced apoptosis observed in PS120 CFTR-transfected cells was related to the activity of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger and to the integrity of CFTR. Therefore, it would be tempting to conclude that lovastatin-induced apoptosis is increased in the presence of CFTR because this protein induces a stimulation of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity, possibly via a cAMP-dependent mechanism. However, the experiments performed in our system do not prove that the mechanism by which CFTR activates the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger during apoptosis involves an elevation of the cAMP levels. These experiments only show that the anion exchanger exhibits an enhanced sensitivity to second messengers in the presence of CFTR, and the exact mechanism of this stimulation will likely be the subject of future studies. Whatever this mechanism is, the loss of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> will lower the pHi, favoring apoptosis by a better activation of endonucleases and a cleavage of DNA. These results provide a molecular mechanism explaining the data of Gottlieb and Dosanjh (14), who demonstrated a role of the functional CFTR in acidification during the initiation of apoptosis in epithelial cells.

In the present study, a basal level of lovastatin-induced apoptosis was observed in all cell lines independent of the expression of CFTR or NHE1. The role of CFTR is, therefore, to increase the ability of the cells to initiate programmed cell death. In PS120 mock-transfected cells, it is surprising to note that cAMP was able to enhance lovastatin-induced apoptosis. Here we show that this effect cannot be attributed to a direct stimulation of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger since in the absence of CFTR, this transporter was not sensitive to the cyclic nucleotide. CFTR could, therefore, act as a molecular adapter mediating the cAMP activation of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. It has been shown that elevated levels of cAMP can induce apoptosis in various cell lines (1, 12, 25, 28, 31). However, in PS120 mock-transfected cells, cAMP was not an apoptotic agent per se since its action had been observed only during lovastatin treatment. Therefore, cAMP could directly activate some of the signaling pathways implicated in lovastatin-induced apoptosis. For example, it has been demonstrated that lovastatin completely inhibits p42/p44 mitogen-activated protein kinase activation by growth factors (F. R. McKenzie, personal communication) and that protein kinase A activation also mediated a partial inhibition of growth factor-stimulated p44 mitogen-activated protein kinase (29).

In conclusion, PS120 cells transfected with human CFTR exhibit an increase in lovastatin-induced apoptosis due to the activation of a Na+-independent Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger leading to a further pHi decrease. The parallel overexpression of the NHE1 isoform completely impairs the ability of CFTR to increase apoptosis by clamping pHi close to neutrality.

Although NHE1 is an extremely efficient acid extruder per se, it is unable to limit the effect of CFTR on apoptosis in CCL-39 cells. Thus this exchanger has to be downregulated during apoptosis. If this hypothesis is true, it will be of great interest to determine whether or not CFTR regulates NHE1. We are currently in the process of investigating this hypothesis.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Poujeol, Unité Mixte de Recherche-Centre National de la Recherche Scientifique 6548, Bâtiment Sciences Naturelles Université de Nice-Sophia Antipolis, Parc Valrose, O6108 Nice Cedex 2, France (E-mail: poujeol{at}unice.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11 December 2000; accepted in final form 30 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aharoni, D, Dantes A, Oren M, and Amsterdam A. cAMP-mediated signals as determinants for apoptosis in primary granulosa cells. Exp Cell Res 218: 271-282, 1995[ISI][Medline].

2.   Akagi, Y, Ito K, and Sawada S. Radiation-induced apoptosis and necrosis in Molt-4 cells: a study of dose-effect relationships and their modification. Int J Radiat Biol 64: 47-56, 1993[ISI][Medline].

3.   Angoli, D, Delia D, and Wanke E. Early cytoplasmic acidification in retinamide-mediated apoptosis of human promyelocytic leukemia cells. Biochem Biophys Res Commun 229: 681-685, 1996[ISI][Medline].

4.   Barasch, J, Kiss B, Prince A, Saiman L, Gruenert D, and al-Awqati Q. Defective acidification of intracellular organelles in cystic fibrosis. Nature 352: 70-73, 1991[ISI][Medline].

5.   Bidet, M, Tauc M, Koechlin N, and Poujeol P. Video microscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules. Pflügers Arch 416: 270-280, 1990[ISI][Medline].

6.   Biwersi, J, and Verkman AS. Cell-permeable fluorescent indicator for cytosolic chloride. Biochemistry 30: 7879-7883, 1991[ISI][Medline].

7.   Boyle, KM, Irwin JP, Humes BR, and Runge SW. Apoptosis in C3H-10T1/2 cells: roles of intracellular pH, protein kinase C, and the Na+/H+ antiporter. J Cell Biochem 67: 231-240, 1997[ISI][Medline].

8.   Choi, JW, and Jung SE. Lovastatin-induced proliferation inhibition and apoptosis in C6 glial cells. J Pharmacol Exp Ther 289: 572-579, 1999[Abstract/Free Full Text].

9.   Clarke, LL, and Harline MC. Dual role of CFTR in cAMP-stimulated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion across murine duodenum. Am J Physiol Gastrointest Liver Physiol 274: G718-G726, 1998[Abstract/Free Full Text].

10.   Earnshaw, WC. Nuclear changes in apoptosis. Curr Opin Cell Biol 7: 337-343, 1995[ISI][Medline].

11.   Franek, F, Vomastek T, and Dolnikova J. Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures. Cytotechnology 9: 117-123, 1992[ISI][Medline].

12.   Gjertsen, BT, Cressey LI, Ruchaud S, Houge G, Lanotte M, and Doskeland SO. Multiple apoptotic death types triggered through activation of separate pathways by cAMP and inhibitors of protein phosphatases in one (IPC leukemia) cell line. J Cell Sci 107: 3363-3377, 1994[Abstract/Free Full Text].

13.   Godard, T, Deslandes E, Lebailly P, Vigreux C, Sichel F, Poul JM, and Gauduchon P. Early detection of staurosporine-induced apoptosis by comet and annexin V assays. Histochem Cell Biol 112: 155-161, 1999[ISI][Medline].

14.   Gottlieb, RA, and Dosanjh A. Cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis. Proc Natl Acad Sci USA 93: 3587-3591, 1996[Abstract/Free Full Text].

15.   Gottlieb, RA, Giesing HA, Zhu JY, Engler RL, and Babior BM. Cell acidification in apoptosis: granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H+-ATPase. Proc Natl Acad Sci USA 92: 5965-5968, 1995[Abstract/Free Full Text].

16.   Gray, MA, Harris A, Coleman L, Greenwell JR, and Argent BE. Two types of chloride channels on duct cells cultured from human fetal pancreas. Am J Physiol Cell Physiol 257: C240-C251, 1989[Abstract/Free Full Text].

17.   Haws, C, Krouse ME, Xia Y, Gruenert DC, and Wine JJ. CFTR channels in immortalized human airway cells. Am J Physiol Lung Cell Mol Physiol 263: L692-L707, 1992[Abstract/Free Full Text].

18.   Huang, P, and Plunkett W. A quantitative assay for fragmented DNA in apoptotic cells. Anal Biochem 207: 163-167, 1992[ISI][Medline].

19.   Illek, B, Yankaskas JR, and Machen TE. cAMP and genistein stimulate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conductance through CFTR in human airway epithelia. Am J Physiol Lung Cell Mol Physiol 272: L752-L761, 1997[Abstract/Free Full Text].

20.   Ishaque, A, and Al-Rubeai M. Use of intracellular pH and annexin-V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture. J Immunol Methods 221: 43-57, 1998[ISI][Medline].

21.   Kim, JA, Kang YS, Lee SH, Lee EH, Yoo BH, and Lee YS. Glibenclamide induces apoptosis through inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels and intracellular Ca2+ release in HepG2 human hepatoblastoma cells. Biochem Biophys Res Commun 261: 682-688, 1999[ISI][Medline].

22.   Lee, MG, Choi JY, Luo X, Strickland E, Thomas PJ, and Muallem S. Cystic fibrosis transmembrane conductance regulator regulates luminal Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in mouse submandibular and pancreatic ducts. J Biol Chem 274: 14670-14677, 1999[Abstract/Free Full Text].

23.   Lee, MG, Wigley WC, Zeng W, Noel LE, Marino CR, Thomas PJ, and Muallem S. Regulation of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells. J Biol Chem 274: 3414-3421, 1999[Abstract/Free Full Text].

24.   Li, J, and Eastman A. Apoptosis in an interleukin-2-dependent cytotoxic T lymphocyte cell line is associated with intracellular acidification. Role of the Na+/H+-antiport. J Biol Chem 270: 3203-3211, 1995[Abstract/Free Full Text].

25.   Lomo, J, Blomhoff HK, Beiske K, Stokke T, and Smeland EB. TGF-beta 1 and cyclic AMP promote apoptosis in resting human B lymphocytes. J Immunol 154: 1634-1643, 1995[Abstract/Free Full Text].

26.   Maiuri, L, Raia V, De Marco G, Coletta S, de Ritis G, Londei M, and Auricchio S. DNA fragmentation is a feature of cystic fibrosis epithelial cells: a disease with inappropriate apoptosis? FEBS Lett 408: 225-231, 1997[ISI][Medline].

27.   Mastrocola, T, Porcelli AM, and Rugolo M. Role of CFTR and anion exchanger in bicarbonate fluxes in C127 cell lines. FEBS Lett 440: 268-272, 1998[ISI][Medline].

28.   McConkey, DJ, Orrenius S, and Jondal M. Agents that elevate cAMP stimulate DNA fragmentation in thymocytes. J Immunol 145: 1227-1230, 1990[Abstract/Free Full Text].

29.   McKenzie, FR, and Pouyssegur J. cAMP-mediated growth inhibition in fibroblasts is not mediated via mitogen-activated protein (MAP) kinase (ERK) inhibition. cAMP-dependent protein kinase induces a temporal shift in growth factor-stimulated MAP kinases. J Biol Chem 271: 13476-13483, 1996[Abstract/Free Full Text].

30.   McNicholas, CM, Guggino WB, Schwiebert EM, Hebert SC, Giebisch G, and Egan ME. Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator. Proc Natl Acad Sci USA 93: 8083-8088, 1996[Abstract/Free Full Text].

31.   Myklebust, JH, Josefsen D, Blomhoff HK, Levy FO, Naderi S, Reed JC, and Smeland EB. Activation of the cAMP signaling pathway increases apoptosis in human B-precursor cells and is associated with downregulation of Mcl-1 expression. J Cell Physiol 180: 71-80, 1999[ISI][Medline].

32.   Perez-Sala, D, Collado-Escobar D, and Mollinedo F. Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease. J Biol Chem 270: 6235-6242, 1995[Abstract/Free Full Text].

33.   Poulsen, JH, Fischer H, Illek B, and Machen TE. Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 91: 5340-5344, 1994[Abstract].

34.   Poulsen, JH, and Machen TE. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent pHi regulation in tracheal epithelial cells. Pflügers Arch 432: 546-554, 1996[ISI][Medline].

35.   Pouyssegur, J. The growth factor-activatable Na+/H+ exchange system: a genetic approach. Trends Biochem Sci 10: 453-455, 1985[ISI].

36.   Pouyssegur, J, Sardet C, Franchi A, L'Allemain G, and Paris S. A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc Natl Acad Sci USA 81: 4833-4837, 1984[Abstract].

37.   Ranasinha, C, Assoufi B, Shak S, Christiansen D, Fuchs H, Empey D, Geddes D, and Hodson M. Efficacy and safety of short-term administration of aerosolised recombinant human DNase I in adults with stable stage cystic fibrosis. Lancet 342: 199-202, 1993[ISI][Medline].

38.   Roos, A, and Boron WF. Intracellular pH. Physiol Rev 61: 296-434, 1981[Free Full Text].

39.   Russ, U, Balser C, Scholz W, Albus U, Lang HJ, Weichert A, Scholkens BA, and Gogelein H. Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes. Pflügers Arch 433: 26-34, 1996[ISI][Medline].

40.   Schroeder, BC, Waldegger S, Fehr S, Bleich M, Warth R, Greger R, and Jentsch TJ. A constitutively open potassium channel formed by KCNQ1 and KCNE3. Nature 403: 196-199, 2000[ISI][Medline].

41.   Schwiebert, EM, Benos DJ, Egan ME, Stutts MJ, and Guggino WB. CFTR is a conductance regulator as well as a chloride channel. Physiol Rev 79: S145-166, 1999[Medline].

42.   Shen, BQ, Widdicombe JH, and Mrsny RJ. Effects of lovastatin on trafficking of cystic fibrosis transmembrane conductance regulator in human tracheal epithelium. J Biol Chem 270: 25102-25106, 1995[Abstract/Free Full Text]. [Corrigenda. J Biol Chem 271: January 1996, p. 1250.]

43.   Stutts, MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, and Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847-850, 1995[ISI][Medline].

44.   Switzer, BR, and Summer GK. A modified fluorometric micromethod for DNA. Clin Chim Acta 32: 203-206, 1971[ISI][Medline].

45.   Thompson, CB. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456-1462, 1995[ISI][Medline].


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