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J. Biol. Chem., Vol. 277, Issue 45, 43041-43049, November 8, 2002

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Reactive Oxygen Nitrogen Species Decrease Cystic Fibrosis Transmembrane Conductance Regulator Expression and cAMP-mediated Cl Secretion in Airway Epithelia^*

Zsuzsa Bebok^§¶, Karoly Varga^§, James K. Hicks^§, Charles J. Venglarik^§**, Timea Kovacs^§, Lan Chen, Karin M. Hardiman, James F. Collawn^§§§, Eric J. Sorscher^§¶, and Sadis Matalon^§**¶¶

From the Departments of  Medicine,  Cell Biology, ^** Environmental Health Sciences,  Anesthesiology, and ^§ The Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama, Birmingham, Alabama 35233

Received for publication, April 2, 2002, and in revised form, August 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We investigated putative mechanisms by which nitric oxide modulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in epithelial cells. Immunoprecipitation followed by Western blotting, as well as immunocytochemical and cell surface biotinylation measurements, showed that incubation of both stably transduced (HeLa) and endogenous CFTR expressing (16HBE14o-, Calu-3, and mouse tracheal epithelial) cells with 100 µM diethylenetriamine NONOate (DETA NONOate) for 24-96 h decreased both intracellular and apical CFTR levels. Calu-3 and mouse tracheal epithelial cells, incubated with DETA NONOate but not with 100 µM 8-bromo-cGMP for 96 h, exhibited reduced cAMP-activated short circuit currents when mounted in Ussing chambers. Exposure of Calu-3 cells to nitric oxide donors resulted in the nitration of a number of proteins including CFTR. Nitration was augmented by proteasome inhibition, suggesting a role for the proteasome in the degradation of nitrated proteins. Our studies demonstrate that levels of nitric oxide that are likely to be encountered in the vicinity of airway cells during inflammation may nitrate CFTR resulting in enhanced degradation and decreased function. Decreased levels and function of normal CFTR may account for some of the cystic fibrosis-like symptoms that occur in chronic inflammatory lung diseases associated with increased NO production.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The contribution of reactive oxygen nitrogen species (RONS)¹ to the development of pathological conditions has been demonstrated for a variety of pulmonary diseases including acute lung injury, asthma, and chronic obstructive pulmonary disease (1-5). Potential sources of nitric oxide (NO) and reactive oxygen species in the airways include activated alveolar and interstitial macrophages (6, 7), neutrophils (8), alveolar type II cells (9, 10), and airway epithelial cells (11). The biological effects of NO depend on its concentration, the chemical nature of the target, and the presence of other radicals (12). NO may bind to the heme group of soluble guanylate cyclase resulting in increased cellular cGMP levels (13), may react with superoxide (O) at diffusion limited rates (6.7 × 10⁹ M¹ s¹) to produce peroxynitrite (ONOO) and higher oxides of nitrogen (12) or, in the presence of an electron acceptor, can react with thiols to form nitrosothiols (14). RONS have been shown to nitrate tyrosine residues in several proteins in vitro and in vivo that may result in their accelerated degradation or loss of function. For example, co-incubation of surfactant protein A (SP-A) with ONOO or tetranitromethane leads to selective nitration of two tyrosines in its carbohydrate-recognition domain and decreases the ability of surfactant protein A to facilitate binding and uptake of Pneumocystis carinii by rat alveolar macrophages (15-17).

The cystic fibrosis transmembrane conductance regulator (CFTR), a 1480-amino acid protein is a member of the traffic ATPase family (18), functions as a cAMP-regulated Cl channel (19), and controls other ion conductive pathways including epithelial chloride (Cl) and sodium (Na⁺) channels, as well as ATP transport (20, 21). Cystic fibrosis (CF) is caused by defective CFTR function (22, 23), and is characterized by abnormal Na⁺ and Cl ion transport in several tissues, including the lungs, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive system (24). In the lungs, airway obstruction by viscous secretions results in chronic inflammation, with acute exacerbations, followed by secondary colonization with Pseudonomas aeruginosa (25). The resultant bronchiectasis and pneumonia leads to pulmonary insufficiency and premature death in more than 90% of CF patients (26).

Interestingly, CF-like symptoms (thickened airway secretions and bronchiectasis) are often seen in patients with lung inflammatory diseases. Previously we have shown that NO, generated by chemical donors, or by fibroblasts stably transfected with iNOS, decreases CFTR expression in transfected LLC-PK₁ cells (27). We therefore hypothesized that post-translational damage to CFTR by RONS may contribute to development of CF-like symptoms in patients with chronic inflammatory disease of the lung without mutations in the CFTR gene. However, the mechanisms involved have not been elucidated. Furthermore, it was unclear if physiological levels of NO and RONS decrease CFTR expression and function in CFTR-expressing airway epithelial cells.

In the present study, we show that NO and RONS decrease wild type CFTR protein levels in airway epithelial cell monolayers, and that this is accompanied by a loss of CFTR function resulting in reduced cAMP-activated Cl currents. The concentration of NO used in our studies was in or below the range measured in vivo during pathologic conditions (3, 28, 29). Decreased CFTR expression results at least in part from nitration of nascent CFTR, and its subsequent degradation by the proteasome. Our results provide the first evidence that NO and RONS can significantly decrease CFTR protein levels and function in epithelial cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Lines-- Calu-3 and 16HBE14o- cells were previously characterized and maintained in the Cystic Fibrosis Research Center at the University of Alabama, Birmingham, AL. Calu-3 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum; 16HBE14o- cells were grown in minimal essential medium with Earle's salt and L-glutamine (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen). HeLa cells expressing wild type CFTR were transduced and selected as described (30). Mouse tracheal epithelial (MTE) cells were isolated from C57BL/6 mice according to a previously described protocol (31). MTE cells were grown in a 1:1 mixture of 3T3 fibroblast preconditioned Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum, 1% penicillin/streptomycin) and Ham's F-12 medium, supplemented with 10 µg/ml insulin, 1 µM hydrocortisone, 3.75 µg/ml endothelial cell growth supplement, 25 ng/ml epidermal growth factor, 30 nM triiodothyronine, 5 µg/ml iron saturated transferrin, and 10 ng/ml cholera toxin. For Ussing chamber experiments, 1 × 10⁶ Calu-3 or MTE cells/filter were seeded onto 6.5-mm diameter Transwell filters (Corning-Costar, Corning, NY), and cultured until the monolayers became confluent.

Antibodies-- The rabbit polyclonal anti-CFTR NBD1 antibody was generated as described (32). The anti-CFTR C-terminal monoclonal antibodies 24-1 and M3A7 were purchased from Genzyme (Cambridge, MA) and Upstate Biotechnology (Lake Placid, NY), respectively. The anti-nitrotyrosine polyclonal antibody was a kind gift from Dr. J. Beckman (University of Alabama at Birmingham), whereas the monoclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Fluorophore-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Molecular Probes (Eugene, OR).

NO Donors-- Human lung epithelial cells 16HBE14o-, Calu-3, and transduced HeLa cells expressing wild type CFTR mounted on filters or plates were treated with 50-200 µM diethylenetriamine NONOate (DETA NONOate; t_1/2 at 37 °C = 20 h; Cayman Chemical, Ann Arbor, MI) for 24-96 h, added into the basolateral compartment. Basolateral media were replaced every 24 h with media containing DETA NONOate. In control studies, cells were incubated either with decayed NO donors or with NO donors and red blood cells as previously described (27). Evolution of NO in the media by DETA NONOate was measured with an ISO-NO electrochemical probe (World Precision Instruments, Sarasota, FL) connected to an IBM-compatible computer via an analog to digital converter. Concentrations of NO in the media were calculated by comparing the signal measured against that was obtained from a NO-saturated water solution that contains 1.94 mM NO.

Preparation of Cell Lysates-- Following exposure of 16HBE14o-, Calu-3, and HeLa cells to DETA NONOate or appropriate control reagents, cells were lysed and total protein was isolated. Equivalent amounts of total protein from each lysate were used for in vitro phosphorylation, immunoprecipitation, and Western blotting assays.

RONS have been shown to nitrate and oxidize proteins (17), and these modifications may cause conformational changes resulting in inhibition of antibody binding or decreased phosphorylation. For this reason, CFTR levels in immunoprecipitates were quantified using either in vitro phosphorylation or Western blotting as described below.

In Vitro Phosphorylation-- CFTR was immunoprecipitated from cell lysates using the anti-CFTR C-terminal antibody 24-1 (Genzyme, Cambridge, MA). Immunoprecipitates were in vitro phosphorylated using the catalytic subunit of the cyclic AMP-dependent protein kinase (Promega, Madison, WI), and [-³²P]ATP (PerkinElmer Life Sciences, Boston, MA) separated by SDS-PAGE, and detected by autoradiography as described (32).

Immunoprecipitation and Western Blotting-- CFTR was immunoprecipitated from cell lysates using the NBD-1 rabbit polyclonal antibody. After being separated by SDS-PAGE through a 6% gel, proteins were transferred to polyvinylidene difluoride membranes (300 mA, 90 min). After blocking with 3% bovine serum albumin, PBS, Tween 20 for 1 h, CFTR bands were detected with the anti-CFTR (M3A7) monoclonal antibody and anti-mouse horseradish peroxidase-conjugated goat IgG, followed by the Super Signal (Pierce, Rockford, IL) enhanced chemiluminescence detection assay.

Detection of CFTR by Western Blotting-- Calu-3 cells grown on 24-well plates were incubated with 100 µM DETA NONOate for 2 or 4 days. At the end of the exposures cells were lysed in 100 µl of RIPA buffer. After centrifugation, total protein concentrations of treated and control samples were normalized, and 30 µl of each cell lysate was mixed with 30 µl of 2× Laemmli sample buffer and subjected to electrophoresis on a 6% gel under reducing conditions. Separated proteins were Western transferred and probed with the anti-CFTR M3A7 monoclonal antibody. The bound primary antibody was detected with anti-mouse horseradish peroxidase-conjugated goat IgG and visualized by chemiluminescence using the Super Signal kit.

Detection of Nitrotyrosine by Western Blotting-- Calu-3 cell lysates (1 × 10⁵ cells/100 µl of lysis buffer) were treated with decreasing concentrations (100, 10, 1, and 0.1 µM) of ONOO a potent protein nitrating agent. After addition of ONOO, samples were centrifuged to remove large protein aggregates and 30-µl samples were mixed with Laemmli sample buffer, warmed at 37 °C for 15 min, and run on a 6% PAGE. Following Western transfer, CFTR was detected using anti-CFTR C-terminal monoclonal antibody and ECL. Membranes were re-used (after stripping in 0.1 M glycine, 1% SDS, 2 M urea) to identify nitrated proteins using an anti-nitrotyrosine polyclonal antibody. In additional experiments CFTR was first immunoprecipitated from cell lysates of Calu-3 cells exposed to ONOO using the 24-1 anti-CFTR antibody. Nitration of immunoprecipitated CFTR was then detected with an anti-nitrotyrosine antibody using Western blotting. Furthermore, the blots were stripped and CFTR was re-probed with a second anti-CFTR antibody (M3A7).

Electrophysiology-- Monolayers of Calu-3 cells were mounted in Ussing chambers (Jim's Instruments, Iowa City, IA), and bathed on both sides with Ringer's solution. Bath solutions were stirred vigorously, gassed with room air, and maintained at 37 °C. Short circuit currents (I_sc) were measured with an epithelial voltage clamp (VCC-600; Physiologic Instruments, San Diego, CA). A 10-mV pulse of 1 s duration was imposed every 10 s to monitor the transepithelial resistance (R_t), which was calculated using Ohm's law. After stabilization of the basal I_sc, the apical solution was changed to a low Cl Ringer's solution to maximize Cl secretion. Calu-3 cells do not express amiloride-sensitive Na⁺ currents. Therefore, 5 min later, when I_sc reached a stable baseline, 50 µM dibutyryl cAMP (Na⁺ salt; Calbiochem, La Jolla, CA) were added into both the apical and basolateral compartments to stimulate Cl secretion. Glybenclamide (100 µM) was used to block the cAMP-induced I_sc, and glybenclamide-inhibitable currents were calculated as an index of Cl movement through CFTR.

Similar electrophysiological measurements were made using MTE cell monolayers mounted in a Ussing chamber. However, in contrast to Calu-3 cells, MTE cells exhibit amiloride-sensitive Na⁺ absorption in addition to Cl secretion. Therefore, Na⁺ absorption was blocked by addition of 10 µM amiloride to the apical bath prior to stimulation of Cl secretion by addition of 10 µM forskolin to both the apical and basolateral baths. Once a new stable baseline was achieved, Cl I_sc was blocked by the addition of bumetanide (100 µM) to the basolateral solution.

Treatment of Cells with 8-Bromo-cGMP-- 100 µM 8-Br-cGMP (a cell permeate analogue of cGMP) was added in the basolateral compartment of Calu-3 cells grown on filters as described above. The medium was removed and fresh medium containing 100 µM 8-Br-cGMP was added daily. After 96 h the cells were mounted in Ussing chambers, and baseline and cAMP-activated Cl I_sc were measured as described above.

Cell Surface Biotinylation-- Cell surface glycoproteins were biotinylated as described (35). Subsequently, CFTR was immunoprecipitated from cell lysates and subjected to SDS-PAGE and Western blot. The biotinylated fraction of CFTR was then detected with horseradish peroxidase-labeled avidin, whereas total CFTR was detected using the anti-CFTR C-terminal monoclonal antibody 24-1. The amount of biotinylated and total CFTR remaining after DETA NONOate treatment was determined by densitometry and plotted as a percentage of that in untreated cells.

Immunocytochemistry-- For nitrotyrosine detection, cells grown on glass coverslips were fixed in 4% formaldehyde solution for 10 min at room temperature. Cells were permeabilized with 0.05% Triton X-100 diluted in PBS, and washed with PBS several times to remove excess detergent. Nonspecific antibody binding sites were blocked by incubating the samples with a 1:20 dilution of normal goat serum in PBS. Anti-nitrotyrosine polyclonal antibody was applied at 1:500 dilution and samples were incubated at least for 6 h or overnight. After subsequent washes in PBS, a secondary, fluorescently labeled antibody was added at 1:400 dilution (AlexaFluor 594, Molecular Probes, Eugene, OR), and incubated in the dark at room temperature for 1 h. Samples were washed and mounted using Vectashield/4,6-diamidino-2-phenylindole mounting medium.

CFTR was detected in ice-cold methanol-fixed cells grown on glass coverslips using either anti-CFTR NBD1 polyclonal antibody or anti-CFTR C-terminal (24-1) monoclonal antibody. In cells grown on permeable supports, CFTR was detected as previously described (33).

Microscopy-- Images were captured on an Olympus IX170 inverted epifluorescence microscope equipped with step motor, filter wheel assembly (Ludl Electronics Products, Hawthorne, NY), and 83,000 filter set (Chroma Technology, Brattleboro, VT). Images were captured with SenSys-cooled charge-coupled high-resolution camera (Photometrics, Tucson, AZ). Partial deconvolution of images was performed using IPLab software (Scanalytics, Fairfax, VA).

Statistical Analysis-- Results are expressed as mean ± S.E. Statistical significance between means was determined by the Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

NO Release by DETA NONOate-- To determine the approximate steady state concentration of NO in the tissue culture medium after addition of 100 µM DETA NONOate (t_1/2 20-22 h), evolution of NO in the medium (pH 7.4, 37 °C) was measured with an ISO-NO electrochemical probe (Fig. 1). Peak NO concentrations were 400-500 nM. Considering the 22-h half-life of DETA NONOate and that fresh DETA NONOate was added to the cells every 24 h, we estimate that the steady state level of NO remained below 400 nM. Furthermore, because DETA NONOate was added into the basolateral compartment, NO concentrations around the apical membranes were probably lower than measured here. Higher NO and nitrate concentrations have been measured in vivo during inflammatory conditions (28). For example, Malinski et al. (34) reported 2-4 µM NO in brain during cerebral ischemia, whereas Gaston et al. (29) reported the presence of 4 µM nitrosothiols in the distal airway fluid of patients with pneumonia. Thus, it seems reasonable to assume that concentrations of NO used in this study are likely to be encountered in the vicinity of airway cells in a number of pathologic situations.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Quantification of NO release from DETA NONOate. DETA NONOate (100 µM) was added to a solution containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum (pH 7.4; T = 37 °C). NO concentration (y axis; nM) was measured continuously with an ISO-NO electrode and plotted versus time (h). Results are of a typical experiment repeated three times.

DETA NONOate Treatment Decreases Steady State CFTR Levels-- The effects of NO and on steady state wild type CFTR levels were tested in Calu-3 and 16HBE14o- cells, which endogenously express wild type CFTR, and in HeLa cells transiently expressing CFTR. For all 3 cell types, co-incubation with 100 µM DETA NONOate, but not with the decayed parent compound, for 48 h resulted in a significant decrease in CFTR levels, as detected by immunoprecipitation followed by either Western blotting (Fig. 2A) or in vitro phosphorylation (Fig. 2B) or Western blotting using the M3A7 monoclonal antibody (Fig. 2C). In addition to this time-dependent decrease in CFTR levels, we found that DETA NONOate also decreased CFTR levels in 16HBE14o- cells in a dose-dependent fashion (Fig. 2D). Exposure of Calu-3 and 16HBE14o- cells to DETA NONOate (50-200 µM) for 1-4 days did not alter total protein levels. Furthermore, the cells appeared morphologically normal when examined by light microscopy, and no significant increase in apoptotic nuclei was detected compared with untreated cells when stained with 4,6-diamidino-2-phenylindole (data not shown). When cells were incubated with previously decayed DETA NONOate or DETA NONOate in the presence of red blood cells (which decrease NO concentrations to nondetectable levels) no significant decrease in CFTR levels could be seen (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   DETA NONOate treatment decreases steady state CFTR levels in transduced HeLa cells, and in epithelial cells (16HBE14o-, and Calu-3) naturally expressing CFTR. A, immunoprecipitation and Western blotting. Cells were incubated with DETA NONOate (100 µM) for 48 h. At that time cells were lysed as described under "Materials and Methods." Cell lysates were immunoprecipitated with anti-CFTR NBD 1 polyclonal antibody, subjected to 6% SDS-PAGE, Western transferred, and detected using the anti-CFTR (M3A7) monoclonal antibody. B, immunoprecipitation and in vitro phosphorylation. Cells were incubated with DETA NONOate (100 µM) for 48 h. At that time cells were lysed as described under "Materials and Methods." In this set of experiments, samples immunoprecipitated with the anti-CFTR (24-1) monoclonal antibody were in vitro phosphorylated using [-32P]ATP and protein kinase A, separated by SDS-PAGE, and detected with autoradiography as described (33). C, Western blotting. Calu-3 cells were treated with 100 µM DETA NONOate for 2 or 4 days. At those time points they were lysed in RIPA buffer containing protease inhibitors and proteins were separated on a 6% PAGE gel and Western blotted. CFTR was probed with anti-CFTR monoclonal antibody (M3A7), and detected with ECL. D, increased concentrations of DETA NONOate decrease CFTR levels in 16HBE14o- cells in a dose-depend- ent manner. 16HBE14o- cells were treated with increasing concentrations (50-200 µM) of DETA NONOate for 24 h, which generated 180 and 800 nM steady state levels of NO, respectively. After lysis, 0.4 mg of total proteins from each sample were immunoprecipitated with anti-CFTR NBD-1 polyclonal antibody and in vitro phosphorylated. E, calnexin levels remained unchanged after DETA NONOate treatment. DETA NONOate-treated and control Calu-3 cells were lysed in RIPA buffer and proteins were separated on 8% SDS-PAGE followed by Western transfer and detection with an anti-calnexin polyclonal antibody. In contrast to CFTR levels (A-D) DETA NONOate treatment had no effect on calnexin levels. In all cases, figures are the results of typical experiments that were repeated at least three times.

To demonstrate that the effect of NO on CFTR was specific and not a result of a global alteration of cell protein expression, we evaluated levels of calnexin (a chaperone protein integral to the endoplasmic reticulum membrane) in untreated and DETA NONOate-treated cell lysates by Western blotting. In contrast to CFTR, calnexin levels remained unchanged after DETA NONOate treatment (Fig. 2E).

DETA NONOate Treatment Significantly Decreases CFTR Function in Calu-3 and MTE Cell Monolayers-- To determine the functional consequences of reduced CFTR expression as a result of NO exposure, Calu-3 (Fig. 3, A and B) or MTE cell monolayers (Fig. 3, C and D) were mounted into Ussing chambers and cAMP-stimulated Cl secretion was measured as described under "Materials and Methods." Addition of 10 µM amiloride into the apical compartments did not alter baseline I_sc values for Calu-3 cells (data not shown). Addition of dibutyryl cAMP (50 µM) to both sides of Calu-3 monolayers resulted in a significant increase in I_sc, which was completely inhibited by glybenclamide (100 µM), indicating that the increase in I_sc was the result of Cl movement through CFTR. When Calu-3 monolayers were co-incubated with 100 µM DETA NONOate for 96 h the basal I_sc or transepithelial resistance did not change (~500-700  cm²) (Fig. 3). However, these monolayers had markedly decreased I_sc values following cAMP stimulation, suggesting a decrease in CFTR-dependent Cl transport. To correlate CFTR protein levels with I_sc measurements, Calu-3 monolayers from Ussing chamber experiments were lysed and CFTR was immunoprecipitated with anti-CFTR NBD1 polyclonal antibody and in vitro phosphorylated using [-³²P]ATP and protein kinase A. As shown in Fig. 4, CFTR protein levels in Calu-3 monolayers with reduced cAMP-activated I_sc were less than 40% of those in nontreated controls. In contrast to CFTR levels, total protein concentrations of DETA NONOate (100 µM)-treated and control cell lysates were similar, indicating that DETA NONOate treatment was not toxic or inhibiting protein synthesis. Furthermore, exposure of Calu-3 cells to 50 µM DETA NONOate for 96 h did not alter cAMP-stimulated I_sc (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   DETA NONOate treatment decreases cAMP-activated Isc in Calu-3 and MTE cells. A, representative tracing shows decreased cAMP-activated Isc in control and DETA NONOate (100 µM for 96 h)-treated Calu-3 monolayers (upper tracing, control (nontreated); lower tracing, DETA NONOate-treated). Arrows indicate the point at which the apical solution was changed to a low Cl solution (6 mM gluconate substitution), the addition of dibutyryl cAMP (50 µM) to both compartments to activate the Cl current, and the addition of glybenclamide (100 µM). B, mean values of glybenclamide-inhibitable currents (Isc) following activation with dibutyryl cAMP in control and DETA NONOate-treated Calu-3 monolayers; values are mean ± 1 S.E., n = 12, p < 0.05. C, representative tracings of untreated (upper tracing) and DETA NONOate-treated MTE cell monolayers (100 µM for 96 h; lower tracing). Amiloride (10 µM; thin arrows) was added in the apical compartments to block Na+ transport, a well known feature of these monolayers. Notice that DETA NONOate-treated monolayers had significantly lower amiloride-sensitive currents. After a new baseline was reached, Cl secretion was stimulated by the addition of forskolin (10 µM) into both compartments followed by bumetanide (100 µM) into the basolateral compartment. D, mean values of bumetanide-inhibitable currents (Isc) following activation with forskolin in control and DETA NONOate-treated MTE cell monolayers; values are mean ± 1 S.E. Summary of Ussing chamber experiments performed in MTE cell monolayers: n = 6, p < 0.05.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   DETA NONOate decreases CFTR protein levels in Calu-3 cells. Calu-3 cell monolayers, tested in Ussing chambers as shown in Fig. 3, were lysed at the end of the experiments. Equal amounts of total proteins were immunoprecipitated with anti-CFTR C-terminal monoclonal antibody, and in vitro phosphorylated using [-32P]ATP and protein kinase A. A, high levels of fully glycosylated (Band C) CFTR in control samples (1-6). Decreased CFTR levels after 4 days treatment with DETA NONOate are shown in lanes 7-12. B, summary of densitometry results shown in panel A. There was a significant decrease of mature (Band C) CFTR in DETA NONOate-treated cells compared with controls. Values are mean ± 1 S.E.; n = 6; *, p < 0.01.

cAMP-activated Cl secretion was also measured in DETA NONOate-treated MTE cells. In contrast to Calu-3 cells, MTE cells exhibit an amiloride-sensitive I_sc resulting from Na⁺ transport. Therefore, amiloride-sensitive currents were blocked before activation of Cl secretion with cAMP. In MTE monolayers both the amiloride-sensitive and cAMP-activated I_sc were decreased as a result of DETA NONOate treatment (Fig. 3, C and D).

8-Bromo-cGMP Has No Effect on CFTR Expression in Calu-3 Cells-- To test whether the effects of NO and RONS on CFTR expression levels were mediated by an increase in cGMP, we treated Calu-3 cells with 8-Br-cGMP and performed Ussing chamber studies followed by immunoprecipitation of CFTR. As shown in Fig. 5, both cAMP-stimulated I_sc and CFTR protein levels were not decreased following exposure to 8-Br-cGMP. Similarly, treatment of Calu-3 cells with 5 µM ODQ (a potent inhibitor of guanylyl cyclase, known to efficiently block the NO-induced accumulation of cGMP (35)) every 12 h did not block the decrease in steady state levels of CFTR induced by treatment with DETA NONOate, indicating that guanylate cyclase activity was not necessary to decrease CFTR levels (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Bromo-cGMP does not decrease cAMP-activated Cl secretion or steady state CFTR levels. 8-Bromo-cGMP (100 µM) was added into the basolateral compartment of Calu-3 cells grown on filters. The medium was removed and fresh medium containing 100 µM 8-Br-cGMP was added daily. After 96 h the cells were mounted in Ussing chambers and cAMP-activated Cl secretion was measured. A, 8-bromo-cGMP treatment had no effect on CFTR function. Glybenclamide-inhibitable cAMP activated Isc following addition of cAMP into monolayers treated with 8-bromo-cGMP or vehicle. Values are mean ± 1 S.E.; n = 4. B, 8-bromo-cGMP treatment had no effect on CFTR protein levels. At the end of the Ussing chamber experiments, CFTR was immunoprecipitated and in vitro phosphorylated to measure changes in CFTR levels. In agreement with the functional data, no changes in steady state CFTR levels as a result of bromo-cGMP treatment could be detected.

DETA NONOate Treatment Decreases CFTR Immunostaining in Epithelial Cell Monolayers-- Changes in the distribution of CFTR as a result of DETA NONOate treatment were examined in both Calu-3 and 16HBE14o- cell monolayers by immunocytochemistry. Co-incubation of Calu-3 cell monolayers with 100 µM DETA NONOate for 96 h and 16HBE14o- monolayers for 24-48 h resulted in decreased CFTR staining (Fig. 6). Both intracellular and surface CFTR levels were decreased. Differences in response times between the two cell lines may reflect differences in CFTR expression (see Fig. 2A), or airway cell versus serous gland cell-specific variations. Furthermore, incubation of either cell line with decayed DETA NONOate did not alter CFTR expression, indicating that the observed effects were not because of the parent compound.

View larger version (77K): [in this window] [in a new window]   Fig. 6.   Decreased CFTR staining in 16HBE14o- and Calu-3 cells after treatment with DETA NONOate. Left panel, 16HBE14o-. Right panel, Calu-3. Cells were cultured on permeable supports. One side view (upper panel) and two top views (middle and lower panels, apical surface and level of nuclei, respectively) are shown. Strong apical CFTR staining is visible in untreated cells. Exposure of 16HBE14o- and Calu-3 cells to 100 µM DETA NONOate for 24 and 96 h, respectively, resulted in decreased CFTR staining both at the apical surface and in the cytoplasm. Results of typical experiments, which were repeated at least three times each. No significant staining was seen when equivalent amounts of nonimmune rabbit IgG was used instead of the primary anti-CFTR antibody.

Changes in Cell Surface and Intracellular Pools of CFTR-- It is possible that NO donors might decrease total cell CFTR protein levels without altering cell surface expression of CFTR. We therefore compared the effects of NO donors on the cell surface and total pools of CFTR in Calu-3 cells by performing cell surface biotinylation combined with immunoprecipitation and Western blotting. A 24-h treatment with DETA NONOate had no significant effect either on the total or the cell surface (biotinylated) fractions of CFTR. However, when cells were treated with NO donors for longer periods (96 h), both intracellular and surface levels were decreased significantly (Fig. 7). These results are in agreement with our functional measurements (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effects of DETA NONOate on total and cell surface CFTR. Both total and cell surface CFTR levels decreased after DETA NONOate treatment. Cell surface biotinylation combined with immunoprecipitation and Western blotting was carried out in control and DETA NONOate-treated Calu-3 cells to detect changes in cell surface and intracellular pools of CFTR. A, total CFTR levels after 24 and 72 h treatment of Calu-3 cells with 100 µM DETA NONOate. CFTR was immunoprecipitated with the anti-NBD1 antibody and detected with the 24-1 antibody on Western blots. Left panel, representative gel. Right panel, summary of densitometry results, plotted as % of control at the corresponding time point. Values are mean ± 1 S.E.; n = 5; *, p < 0.002. B, changes in the cell surface pools of CFTR after 24 and 72 h treatment with 100 µM DETA NONOate. After cell surface biotinylation, immunoprecipitation with anti-CFTR NBD1 polyclonal antibody, 6% SDS-PAGE, and Western blotting the biotinylated (cell surface) fraction of CFTR was detected with avidin horseradish peroxidase. Left panel, representative gel showing a decrease in cell surface CFTR levels. Right panel, summary of densitometry results, plotted as % of control at the corresponding time point. Values are mean ± 1 S.E.; n = 5; *, p < 0.002.

CFTR Nitration Promotes Proteasomal Degradation-- Previous studies have demonstrated that RONS are capable of nitrating proteins in vitro (36). Furthermore, loss of function and/or enhanced proteasomal degradation of nitrated proteins have been shown (37-39). Based on these observations, we tested the hypothesis that RONS nitrate tyrosine residues in CFTR, and that this modification enhances proteasomal degradation of CFTR. DETA NONOate treatment increased nitrotyrosine immunostaining in all cell lines tested (HeLa, Calu-3, and 16HBE14o-) (Fig. 8). Nitrotyrosine localized mainly to the perinuclear region. In the presence of 50 µM ALLN (for 12 h) and NO donors, increased cell staining for both CFTR and nitrotyrosine was found. In addition, nitrotyrosine immunostaining was detected at the cell surface in ALLN-treated cells (B, right panel) in a pattern similar to that of cell surface CFTR in nonpolarized 16HBE cells (upper panel). These results indicate that incubation of cells with NO donors results in protein nitration, and that nitrated proteins are degraded by the proteasome. We were unable to co-localize CFTR and nitrotyrosine in these cells because conditions to achieve optimal staining were different for the two antibodies. Therefore, specific nitration of CFTR was shown by biochemical methods (see below).

View larger version (105K): [in this window] [in a new window]   Fig. 8.   DETA NONOate increases intracellular and cell surface nitrotyrosine staining in 16HBE14o- cells. A, CFTR immunostaining in 16HBE cells grown on glass coverslips. B, increased perinuclear nitrotyrosine staining in DETA NONOate (100 µM)-treated cells, which is further enhanced and accompanied by peripheral and surface staining, in the presence of proteasome blockade with ALLN. Control: untreated cells; DETA NONOate, exposure to DETA NONOate (100 µM) for 48 h; DETA+ALLN, exposure to DETA NONOate (100 µM, 48 h) and ALLN (50 µM, 10 h). Green, CFTR; red, nitrotyrosine; blue, nuclear staining.

Biochemical Detection of Nitrotyrosine in CFTR-- The immunocytochemical results presented above point to the involvement of the proteasome in the degradation of nitrated CFTR and other proteins.

To confirm that CFTR is a target for nitration by RONS, we exposed Calu-3 cell lysates to varying concentrations (100, 10, 1, and 0.1 µM) of authentic ONOO for 5 min. After centrifugation to remove protein aggregates, proteins were separated by SDS-PAGE, and Western blots were transferred. Blots were first probed with anti-CFTR C-terminal monoclonal antibody to identify CFTR. As shown in Fig. 9A, higher concentrations of ONOO caused protein aggregation in the lysates, resulting in decreased CFTR levels (lanes 2 and 3). After identification of the CFTR bands, membranes were stripped and nitrated proteins were detected using an anti-nitrotyrosine monoclonal antibody. Several proteins were effectively nitrated when lysates were treated with 100 µM ONOO resulting in a smear on the blots. However, when lysates were treated with 10 µM ONOO only a few proteins were nitrated. Most importantly, the previously identified CFTR band (using anti-CFTR antibody) was also stained using the anti-nitrotyrosine antibody. These results further support our hypothesis that CFTR is a target for nitration by RONS.

View larger version (42K): [in this window] [in a new window]   Fig. 9.   Identification of nitrated CFTR in ONOO-treated Calu-3 cell lysates. A, Calu-3 cell lysates were treated with ONOO (100, 10, 1, and 0.1 µM), proteins were separated on 6% PAGE and Western transferred. CFTR was detected with anti-CFTR monoclonal antibody (24-1, left panel) and after stripping, nitrotyrosine was detected with anti-nitrotyrosine antibody (right panel). CFTR was identified as one of the nitrated proteins based on molecular weight, shape of the band, and reactivity with both anti-CFTR and anti-nitrotyrosine antibodies. Results are of a typical experiment repeated three times. B, ONOO-treated and control Calu-3 cell lysates were immunoprecipitated with anti-CFTR (24-1) antibody (IP, a-CFTR), run on a 6% PAGE, and detected with anti-nitrotyrosine antibody (left panel). Only the ONOO-treated sample (+) stained positive with anti-nitrotyrosine antibody. After stripping, CFTR was re-probed with anti-CFTR M3A7 antibody to show the presence of CFTR bands both in the control and ONOO-treated samples (+ and ).

To provide additional evidence for the presence of nitrated CFTR, ONOO-treated and control Calu-3 cell lysates were immunoprecipitated with anti-CFTR 24-1 antibody, separated by SDS-PAGE, and probed with anti-nitrotyrosine antibody (Fig. 9B). Nitrotyrosine was only detected in the ONOO-treated sample. After stripping, CFTR was re-probed with anti-CFTR M3A7 antibody to show the presence of CFTR bands in both the control and ONOO-treated samples.

	DISCUSSION

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Several studies have provided evidence that nitration reactions occur in vivo during inflammatory processes. 3-Nitrotyrosine residues, products of the addition of a nitro group (NO₂) to the ortho position of the hydroxyl group of tyrosine, are stable end products which therefore, serve as footprints of RONS action. They are readily detectable by immunohistochemistry, enzyme-linked immunosorbent assay, or high pressure liquid chromatography (40) and are commonly detected in tissues infiltrated by neutrophils and monocytes during infectious and inflammatory processes (1, 2). In vitro, proteins can be nitrated either by ONOO or by reactive intermediates generated by the myeloperoxidase-catalyzed reaction of reactive species released from activated neutrophils (41, 42).

Protein nitration and oxidation by RONS in vitro have been associated with diminished function of a variety of crucial proteins present in the alveolar space, including surfactant protein A and ₁-proteinase inhibitor (16, 43). Gole et al. (44) also reported the presence of nitrated ceruloplasmin, transferrin, ₁-protease inhibitor, ₁-anti-chymotrypsin, and -chain fibrinogen in the plasma of patients with ALI/ARDS. Using quantitative enzyme-linked immunosorbent assay and high pressure liquid chromatography, we detected levels of protein-associated nitrotyrosine in the epithelial lining fluid of patients with acute lung injury and hydrostatic edema (3) that were at least an order of magnitude higher than those found in proteins in normal human BAL fluid (28 pmol/mg of protein) (45).

It is well accepted that under inflammatory conditions, both infiltrating cells (granulocytes, macrophages) and respiratory epithelial cells produce high levels of NO. Conditions necessary for the formation of the reactive species and nitration or oxidation of respiratory epithelial cell proteins are therefore present in the inflamed lung (12). Our experiments support the hypothesis that wild type CFTR levels and function can be decreased by reactive species in vivo. We used a highly controlled system (human lung epithelial cell monolayers cultured in vitro) exposed to chemical NO donors liberating NO at a concentration that can easily be reached in vivo (~500 nM) to investigate the mechanism by which NO and reactive species modify CFTR. We found that NO donor treatment causes a significant decrease in CFTR protein levels in endogenous CFTR expressing human epithelial cells without causing either significant changes in total protein concentrations or cell toxicity. Decreased protein levels were accompanied by reduced CFTR function as judged by cAMP-activated Cl current measurements in Ussing chambers. Moreover, whereas 24 h of treatment with NO donors was sufficient to cause a significant decrease in CFTR expression in 16HBE14o- cells (which express low levels of CFTR representative of those in airway surface epithelium), a longer period (3-4 days) of NO treatment was necessary to induce a significant decrease in CFTR expression and function in Calu-3 cells (a human lung, submucosal serous gland cell line that express very high levels of CFTR). These results suggest that the severity of damage to CFTR in vivo caused by the reactive species may differ between cells in the airway epithelium (16HBE14o-) and those in submucosal glands (Calu-3). It is important to note that in neither case did exposure to RONS result in general cytotoxicity because the integrity of the monolayers and the morphology of the cells tested did not change as a result of NO donor treatment.

There are several mechanisms by which RONS may modulate expression and function of CFTR in the respiratory epithelium. Our experiments, in which proteins in cell lysates were directly nitrated using authentic ONOO (Fig. 9), indicate that CFTR is very effectively nitrated. Furthermore, the effects of RONS are not cell line or expression system specific. This is an important finding because while prior studies have demonstrated a significant effect of NO and reactive species on transgene expression after gene transfer (27), similar effects on endogenous CFTR Cl channel function have not been described to date.

One possible mechanism by which RONS decrease the steady state levels of CFTR is by inhibition of CFTR maturation. Although we have not performed pulse-chase studies in airway cells, previous studies have shown that exposure of HeLa cells, stably transduced with CFTR to 50-100 µM DETA NONOate for 4 h resulted in maturation inhibition of CFTR (46). Whether long term exposure of airway cells to DETA NONOate also leads to maturation inhibition will need to be addressed in future studies. Because RONS simultaneously increased the level of nitrated proteins, including CFTR, in these cells, inhibition of CFTR maturation may be a direct consequence of nitration and degradation of CFTR. This hypothesis is supported by our finding of a further increase in levels of nitrated CFTR in the presence of a proteasome inhibitor, an observation confirmed by immunocytochemistry. This finding indicates that like nitrated -synuclein, nitrated CFTR may be degraded by the proteasome (38). On the other hand, in Saccharomyces cerevisiae ONOO was found to be more potent than hydrogen peroxide in oxidizing thiols, inducing heat shock proteins (Hsp70), and enhancing ubiquitination of proteins (47). Because Hsp70 facilitates endoplasmic reticulum-associated degradation of CFTR in yeast, this mechanism could also account for the decreased CFTR levels we see in our studies (48). Another possibility is that inhibition of CFTR maturation is at least partly a consequence of nitration of chaperone proteins themselves. In Escherichia coli, nitration of the Hsp70 co-chaperone DnaJ (47) results in DnaJ misfolding and loss of function. Because the Hdj-2/Hsc70 chaperone pair is necessary for CFTR biogenesis (49), it is possible that nitration and loss of function of co-chaperone proteins may also inhibit CFTR biogenesis.

It is important to stress that our data do not imply that nitration is the only mechanism responsible for the decrease in CFTR levels and function. In several systems, the biological effects of NO on transport proteins have been associated with the formation of nitrosothiols (50-53). Although it should be stressed that the direct reaction of NO with thiols is unfavorable, the presence of strong electron acceptors, such as Fe³⁺ and oxygen, in biological systems facilitates this reaction through the formation of the nitrosonium ion (NO⁺) intermediate. Once formed, nitrosothiol adducts stabilize NO and may decrease its cytotoxic potential while maintaining or promoting its bioactivity. This appears to be the instance in neurons where NO donors generating NO⁺, but not NO per se, resulted in an S-nitrosylation of critical thiols at the receptor for the N-methyl-D-aspartic acid redox modulatory site. This subsequently prevents excess Ca²⁺ entry into cells and reduced the neurotoxcity associated with NO (51). In contrast, formation of S-nitrosoglutathione in the corpus cavernosum smooth muscle stabilizes the bioactivity of NO resulting in an up-regulation of the Na,K-ATPase activity and subsequent muscle relaxation (50).

There is convincing evidence that NO can also modulate cation channel activity by increasing cGMP. Light et al. (54) demonstrated the presence of a 28-picosiemen cation channel in rat renal inner-medullary collecting duct cells, the activity of which was modulated both by cGMP per se and via PKG-induced phosphorylation. Subsequent studies on cultured collecting duct cells demonstrated that NO released from endothelial cells specifically inhibited the apical membrane Na⁺ conductance in permeabilized monolayers (55). These findings are consistent with a NO-mediated inhibition of Na⁺ reabsorption leading to an increased urinary Na⁺ excretion (54, 55). More recently Jain et al. (56) reported that NO suppressed the activity of a cation channel in the apical membrane of freshly isolated alveolar type II cells through a cGMP-dependent protein kinase pathway. Furthermore, NO donors (either spermine or PAPA NONOate) added into the apical compartment of Ussing chambers, containing cultured alveolar type II cells grown into confluent monolayers, also inhibited vectorial Na⁺ transport by inhibiting both apical Na⁺ entry pathways and Na,K-ATPase (57). However, in this case the effect of NO was cGMP-independent. Finally, intratracheally instillation of DETA NONOate into the alveolar space of rabbits decreased amiloride-sensitive fluid clearance (58).

Because of these findings we investigated whether the effects of NO on CFTR expression and function were mediated by cGMP. We found that incubation of Calu-3 cells with cGMP for 96 h did not alter cAMP-stimulated Cl currents or CFTR function. Similarly, when cells were treated with ODQ (a specific blocker of guanylate cyclase) and DETA NONOate, steady state CFTR levels decreased to the same extent as in cells treated with DETA NONOate alone, indicating that guanylate cyclase activity was not necessary to decrease CFTR levels. These findings are in agreement with our previous report that ^·NO impairs the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms (27).

Taken together, our experiments provide the first evidence that CFTR expression and function can be affected by RONS in vitro, indicating that a similar process may take place in vivo under inflammatory conditions. These negative effects on CFTR function could potentially contribute to CF-like symptoms in a variety of inflammatory airway diseases. Our results provide support for studies investigating CFTR function in inflammatory lung diseases other than cystic fibrosis.

	ACKNOWLEDGEMENTS

We acknowledge Drs. J. S. Hong, J. P. Clancy, and I. Davis for helpful suggestions and critical reading of the manuscript and Dr. John Crow with assistance in measuring NO release by DETA and PAPA NONOate.

	FOOTNOTES

^* 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.

^¶ Supported by CFF Grant R464 and National Institutes of Health NIDDK Grants P30 DK54781 and P50 DK53090.

^§§ Supported by CFF Grant COLLAW00G0.

^¶¶ Supported in part by National Institutes of Health Grants HL31197, HL51173, and P30 DK54781. To whom correspondence should be addressed: Dept. of Anesthesiology, University of Alabama, 901 19th St. S, BMR II, Rm. 224, Birmingham, AL 35205-3703. Tel.: 205-934-4231; Fax: 205-934-7437; E-mail: Sadis.Matalon@ccc.uab.edu.

Published, JBC Papers in Press, August 22, 2002, DOI 10.1074/jbc.M203154200

	ABBREVIATIONS

The abbreviations used are: RONS, reactive oxygen nitrogen species; NO, nitric oxide; CFTR, cystic fibrosis transmembrane conductance regulator; MTE, mouse tracheal epithelial; ONOO, peroxynitrite; PBS, phosphate-buffered saline; DETA, diethylenetriamine.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES