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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 553-560, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4843

Cystic Fibrosis Transmembrane Conductance Regulator Modulates Neurosecretory Function in Pulmonary Neuroendocrine Cell-Related Tumor Cell Line Models

Jie Pan, Christine Bear, Susan Farragher, Ernest Cutz and Herman Yeger

Department of Paediatric Laboratory Medicine and Programme in Structural Biology and Biochemistry, Research Institute, The Hospital for Sick Children; and Departments of Laboratory Medicine & Pathobiology and Physiology, Faculty of Medicine, University of Toronto, Toronto, Canada

Address correspondence to: Herman Yeger, Ph.D., Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8 Canada. E-mail: hermie{at}sickkids.on.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pulmonary neuroendocrine cell (PNEC) system consists of solitary cells and distinctive cell clusters termed neuroepithelial bodies (NEB) localized in the airway epithelium. PNEC/NEB express a variety of bioactive substances, including amine (serotonin, 5HT) and neuropeptides. We have previously shown that NEB cells are O2 sensors expressing nicotinamide adenine diphosphate oxidase complex and O2 sensitive K+ channel. Recently, we demonstrated expression of functional cystic fibrosis transmembrane conductance regulator (CFTR) and Cl- conductances in NEB cells of rabbit neonatal lung. Because PNEC/NEB are sparsely distributed and difficult to study in native lung, we investigated small-cell lung carcinoma (SCLC) and carcinoid tumor cell lines (tumor counterparts of normal PNEC/NEB) as models for PNEC/NEB. SCLC (H146, H345) and carcinoid (H727) cell lines express neuroendocrine cell markers, including chromogranin A, neural cell adhesion molecule (N-CAM), 5HT, and tryptophan hydroxylase. We report that H146, H345, and H727 express CFTR messenger RNA (reverse transcription polymerase chain reaction) and protein (immunoblotting) and possess functional CFTR Cl- conductance, demonstrated by an iodide efflux assay inhibitable by transfection with antisense CFTR. Using an immunoassay to quantitate 5HT secretion, we also show that downregulation of CFTR abolishes hypoxia-induced 5HT release, and reduces secretory response to high potassium. Our findings suggest that CFTR may modulate neurosecretory activity of PNEC/NEB possessing O2 sensor function. We propose that these tumor cell lines may be useful models for investigating the role of CFTR in PNEC/NEB functions in health and disease.

Abbreviations: cystic fibrosis pancreatic adenocarcinoma, CFPAC-1 • cystic fibrosis transmembrane conductance regulator, CFTR • neural cell adhesion molecule, N-CAN • neuroepithelial body, NEB • pulmonary neuroendocrine cells, PNEC • reverse transcription polymerase chain reaction, RT-PCR • small-cell lung carcinoma, SCLC • tryptophan hydroxylase, TH


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The multifunctional pulmonary neuroendocrine cell (PNEC) system, consisting of solitary neuroendocrine cells and innervated PNEC clusters, termed neuroepithelial cell bodies (NEB), have recently been shown to play critical roles in lung development, O2 sensing, and regeneration of distal pulmonary epithelium (1–4). In a rabbit lung model, we have demonstrated that NEB cells express functional cystic fibrosis transmembrane conductance regulator (CFTR) and Cl- conductances during the neonatal period corresponding to peak NEB numbers and the transition to air breathing (2, 5). It is now known that CFTR regulates other ion channels including pulmonary epithelial cell Na+ channels (6–13). Since the O2 sensing complex in NEB involves O2-sensitive K+ channels (1, 14, 15), we have postulated that the function of CFTR in PNEC/NEB could be physiologically important. However, a significant limitation to more detailed biochemical and molecular investigations of this relationship is the relative paucity of PNEC/NEB cells, which represent < 1% of the lung parenchyma.

An alternate model to native PNEC/NEB are immortalized cell lines of small-cell lung carcinomas (SCLC) or variant carcinoid tumor which represent the tumor cell counterpart to PNEC/NEB. Although not entirely representative, these tumor lines recapitulate neuroendocrine cell phenotype, including the ability to synthesize bioactive amine (serotonin, 5HT) and a variety of neuropeptides (16–19), as wells as O2 sensing properties (20). In this study, we analyzed SCLC cell lines H146 and H345 and carcinoid cell line H727 for coordinate expression of CFTR and neuroendocrine cell markers. These cell lines have been previously characterized and shown to be representative models of the PNEC system (21, 22). Similar to the O2 sensory function present in NEB (1), the H146 cell line has been shown to express a functional nicotinamide adenine diphosphate oxidase complex and a hypoxia-sensitive K+ channel function; in addition, H146 cells express K+ channels related to the tandem P domains in a weak inwardly rectifying K+ channel–related acid sensitive K+ channel (20). We report that H146, H345, and H727 express functional CFTR coordinately with expression of 5HT and neuroendocrine cell phenotype using reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. To verify expression of functional CFTR CL- channel conductances, an efflux assay and an antisense CFTR approach were used. To quantitate 5HT release under stimulus-induced secretion in the presence and absence of CFTR Cl- conductance, we used a sensitive competitive ELISA for 5HT. Our studies expand upon the known characteristics of SCLC and carcinoid cell lines and their usefulness for biochemical, electrophysiologic, and molecular studies on PNEC/NEB function, especially in relation to CFTR and its role in CF lung disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines
The classical SCLC cell lines, H345 and H146, and carcinoid cell line H727 (a more differentiated phenotype related to classical SCLC) were obtained from ATCC (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium medium supplemented with 10% FBS. While H727 cells grow attached to the substratum, H345 and H146 cells grow in suspension and require a collagen (rat tail or Vitrogen; Sigma, St. Louis, MO) or poly-L-lysine (Sigma) (0.1% solution in water)-coated substratum for attachment. All other biochemicals used in the study were obtained from Sigma. For immunofluorescence labeling and CFTR functional assays, the cells were cultured on round glass coverslips in multiwell dishes, in 35 mm dishes, and in T25 flasks. As positive or negative controls for the different assays, other cell lines used in the study included colon carcinoma T84, with functional CFTR, cystic fibrosis pancreatic adenocarcinoma (CFPAC-1) representing a non-functional CFTR cell line, and a normal human lung fibroblast (NHLF) non-CFTR expressing cell line; all were maintained in Dulbecco's modified Eagle's medium supplemented with FBS.

Biochemical and Molecular Analysis of Neuroendocrine and CFTR Expression
Expression of the neuroendocrine-specific marker, tryptophan hydroxylase (TH), the rate-limiting enzyme for serotonin synthesis, was determined by RT-PCR using PCR primers and protocols as previously described (23). CFTR expression was determined by immunoblotting after immunoprecipitation with anti-CFTR antibody and by RT-PCR as previously described (23). Actin and {alpha}-tubulin were used to verify equal complementary DNA (cDNA) loading and PCR conditions. Controls included RNA preparations from T84, which expresses intact CFTR messenger RNA (mRNA), and lung fibroblasts, which are negative for both CFTR and neuroendocrine markers.

Immunofluorescence Labeling and Confocal Microscopy
Dual immunofluorescence labeling and confocal laser microscopy were used to demonstrate the coexpression of neuroendocrine cell markers (chromogranin A, N-CAM, serotonin or 5HT) with CFTR. Antibodies used included, polyclonal antibody against 5HT (Diasorin, Stillwater, MN), monoclonal antibody against human chromogranin A (Dako Diagnostic, Mississauga, ON), N-CAM (Euro-Diagnostica, Medeon, Sweden), and the anti-CFTR monoclonals M24–1 and TAM-18 (R&D Systems, Minneapolis, MN and Neomarkers, Fremont, CA, respectively) and MATG1031 and MATG1061 (Transgene, Strasbourg, France) as previously used in our studies of CFTR in PNEC/NEB (23). Antibodies were applied to cells after fixation in 4% paraformaldehyde, with or without permeabilization, and labeling detected by indirect immunofluorescence or after catalyzed reporter deposition amplification as in the case of CFTR (23). For nuclear counterstaining, Sytox Green (Molecular Probes, Eugene, OR) was applied at 1/100,000 dilution for 2 min after immunolabeling.

Iodide Efflux Assay for Determination of CFTR Cl- Conductance
An iodide efflux assay (24) was modified and used to demonstrate Cl- channel secretory function as previously described (25). Cell lines were grown in 35 mm dishes and loaded with iodide by incubation in 136 mM NaI, replacing NaNO3 in the efflux buffer that was at a pH of 7.4 and contained 136 mM NaNO3, 4 mM KNO3, 2 mM Ca (NO3)2, 2 mM Mg(NO3)2, 20 mM Hepes, and 11 mM glucose. Baseline (unstimulated) Cl- secretion was first obtained by collection of 1 ml fractions for 5 min before stimulation of cultures with agonists followed by collection of 1 ml fractions over the next 15 min. Iodide secretion was detected with an iodide-sensitive electrode (Orion Research Inc., Beverly, MA) and pH meter. At either 13 min or 15 min poststimulation, cells were lysed with 0.5% Triton X-100 (Sigma, St. Louis, MO) for 2 min to obtain cellular iodide content. Iodide efflux followed a typical decay curve unless CFTR was stimulated with a CFTR-specific cyclic adenosine monophosphate (cAMP) agonist cocktail (20 µM forskolin, 200 uM cytidine triphosphate–cAMP, 200uM 3-isobutyl-1-methylxanthine). Iodide equivalent to chloride concentration was calculated (25) and plotted graphically over time. Values represent triplicates with standard errors calculated. Statistical significance was determined with the Mann-Whitney test (significance at P < .05).

Pharmacologic Stimulation and Antisense Inhibition of CFTR Function
To verify the identity of CFTR protein expression shown by immunofluorescence labeling, RT-PCR, and Cl- efflux, cells were transfected with antisense CFTR cDNA to downregulate CFTR expression and then assayed for Cl- secretion. Sense cDNA of CFTR representing the first 1.4 Kb of the coding region (26) was reverse-cloned into the plasmid cDNA 3.1(+) vector. The efficacy and specificity of the antisense construct was assessed on Caco2 cells that endogenously express CFTR protein. Western blot analysis was performed on Caco2 protein extracts using the CFTR monoclonal antibody M3A7 previously described (27). The CFTR-specific band was significantly decreased by antisense transfection (Choudhury and Bear, unpublished). H727 cells were transfected with antisense CFTR using SuperShuttle 20 (Quantum Biotechnologies, Inc., Montreal, PQ) as per manufacturer's instructions, and stable transfectants selected under G418. These stably transfected cells were then expanded and assessed with the iodide efflux assay for CFTR Cl- conductance in the presence of cAMP agonists.

Measurement of 5HT Secretion by ELISA: Effects of Hypoxia and High Potassium
We have previously documented 5HT secretion in cultures of NEB isolated from rabbit fetal lungs and SCLC cell line H69 using HPLC method (28). In this study, a sensitive competitive ELISA method (IBL, Hamburg, Germany) was used. Cultures (2 x 104 cells in 35 mm dishes, in triplicate) of H146 and the stable H146 CFTR- antisense transfectant were first rinsed and incubated for 5 min in CO2-independent medium (GibcoBRL, Burlington, ON, Canada) supplemented with 1.2% glucose. Basal secretion under normoxic conditions (5% CO2, 20% O2) was measured after 60 min incubation at 37°C in 1 ml of bicarbonate–buffer salt solution. Cells were resuspended with the preincubated bicarbonate–buffer salt solution at pH 7.4: NaCl, 116 mM; KCl, 5 mM; NaHCO3, 24 mM; CaCl2, 2 mM; MgCl2, 1.1 mM; HEPES, 10 mM; glucose, 5.5mM; and 50 µM pargyline (to prevent 5HT re-uptake) was added freshly. Supernatants were collected after exposure of cultures for 60 min to hypoxia (5% O2/5% CO2/90% N2). To measure 5HT secretion using high extracellular K+ conditions (30 mM K+), 25 mM NaCl was replaced by equimolar KCl in the bicarbonate–buffer salt solution. Samples were collected after 60 min incubation at 37°C under normoxic conditions. All collected samples were lyophilized under vacuum and then stored at -80°C until assayed. To quantitate 5HT content, the lyophilized samples were reconstituted in 100 µL of distilled water and assayed by a commercially available ELISA (IBL) (29). Briefly, the assay was performed as follows: samples and 5HT standard dilutions were applied to 96-well microtiter plates previously coated with goat antirabbit antibody followed by 50 µl biotin-labeled 5HT and 50 µl rabbit antibody against 5HT. After incubation overnight at 4°C and washing with PBS, 150 µl goat antibiotin antibody alkaline phosphatase conjugate in Tris buffer was incubated for 2 h at room temperature with gentle mixing and was washed again; p-nitrophenylphosphate substrate was added, and the enzyme reaction was terminated after 60 min by addition of 50 µl of 1 M sodium hydroxide. Absorption was measured at 405 nm using a microtiter plate reader (Versamax; Molecular Devices, Sunnyvale, CA), and the concentration of 5HT was calculated from the reference curve using the SOFTmax PRO version 3.0 software program (Molecular Devices). As negative and positive controls, respectively, T84 and the SCLC H69 cell line were run in all experiments. The nonparametric, unpaired t test was used for comparison between two groups with Prism GraphPad Software version 2.01 (GraphPad Software Inc., San Diego, CA). P values of < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunolocalization of Neuroendocrine Markers and CFTR in SCLC Cell Lines
Immunofluorescence labeling of the cell lines clearly demonstrated that H345, H146, and H727 expressed significant levels of 5HT, chromogranin A, and N-CAM (Figures 1F–1H, 1K–1M, 1Q, and 1R). In contrast, and as expected, the T84 cell line, used as a negative control, did not express any of these neuroendocrine markers (Figures 1A–1C). The SCLC and carcinoid cell lines showed strong immunoreactivity for N-CAM specifically localized to the plasma membrane. The cobblestone pattern of immunostaining suggested strong intercellular adhesion. In contrast, both 5HT and chromogranin A immunoreactivity was localized in the cytoplasm (Figures 1F, 1G, 1K, 1L, 1P, and 1Q).



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  		Figure 1. Immunofluorescence labeling evidence for expression of neuroendocrine markers 5HT (A, F, K, P), chromogranin A (B, G, L, Q), N-CAM (C, H, M, R) and CFTR, demonstrated by two anti-CFTR antibodies, M24–1 (D, I, N, S) and TAM18 (E, J, O, T) monoclonal antibodies in SCLC cell lines H146 (K–O) and H345 (P–T) and carcinoid cell line H727 (F–J). The control tumor cell line T84 (A–E) shows no expression of neuroendocrine markers but expresses CFTR as expected. NHLF (negative control) do not stain with anti-CFTR antibodies (U, V).

 
In comparison to the neuroendocrine cell markers, CFTR protein expression was clearly defined with both the M24–1 anti-CFTR monoclonal antibody, which recognizing the c-terminus, and TAM-18 anti-CFTR monoclonal antibody, which recognizes the nucleotide binding domain 2, with varying staining intensity (Figures 1D, 1E, 1I, 1J, 1 N, 1O, and 1T). As expected, the T84 cell line showed strong immunoreactivity for CFTR protein with prominent localization to the plasma membrane (Figures 1D and 1E). A similar staining pattern was noted in the carcinoid and SCLC cell lines, with H146 exhibiting greater variability in that both plasma membrane and cytoplasmic staining was observed (Figures 1N and 1O). Control NHLF did not stain with any anti-CFTR antibodies (Figures 1U and 1V). To demonstrate transmembrane localization and insertion of CFTR protein, MATG1031 monoclonal antibody, which recognizes an epitope on the extracellular loop of CFTR, was applied as previously described using catalyzed reporter deposition amplification and confocal microscopy (23) (Figure 2A). In H146 cells, expression of CFTR at the plasma membrane was nonhomogeneous, with potential clustering of CFTR in localized regions of the plasma membrane (Figure 2A). The anti-CFTR MATG1061 monoclonal antibody, which recognizes an internal epitope on the NBD1 domain of CFTR, showed intracytoplasmic and membrane distribution of CFTR protein in H146 cells, as demonstrated by confocal microscopy (Figure 2B).



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  		Figure 2. (A) The H146 SCLC cell line examined by confocal laser microscopy (x63 objective) after labeling for an epitope on the external loop of CFTR using the MATG-1031 monoclonal antibody. H146 cells were kept on ice and immunostained without fixation and permeabilization to restrict access of the antibody only to CFTR exposed on the plasma membrane. Catalyzed reporter deposition amplification was used to enhance detection. The labeling pattern shows isolated sites and clustering of CFTR protein. (B) H146 cells fixed in zinc formalin and permeabilized with 0.5% Triton X-100 were immunolabeled with MATG-1061, which recognizes an internal epitope on the NBD1 domain of CFTR, and examined by confocal microscopy. There is obvious localization of CFTR in both intracytoplasmic and membrane domains. Nuclei were counterstained with Sytox Green and the image was captured and rendered in red color.

 
SCLC and Carcinoid Cell Lines Express mRNAs for TH and CFTR
Western blot analysis of immunoprecipitated CFTR protein confirmed the expression of relatively large amounts of CFTR protein (seen as a doublet) in extracts of T84 cells and comparable or lower amounts in SCLC cell lines H146, H345, and carcinoid cell line H727, respectively (Figure 3). Higher molecular weight forms of CFTR were noted in H146 and H345 cells, most likely representing glycosylation variants. NHLF were negative for CFTR. RT-PCR analysis confirmed the expression of CFTR in the respective tumor cell lines (Figure 3). The expected PCR product was obtained for T84 and the carcinoid and SCLC cell lines. As expected, no PCR product for CFTR was seen with lung fibroblasts. RT-PCR analysis for the neuroendocrine marker, TH, showed strong signals in SCLC cell lines H146, H345, and H727 carcinoid cell line, confirming that these cell lines synthesize 5HT. As expected, both the T84 cell line and lung fibroblasts were negative for TH message. The actin and tubulin PCR controls demonstrated equivalent amounts of cDNA were used in the different PCR reactions. Finally, although not apparent by immunocytochemistry, the discrepancy between the amount of CFTR mRNA and protein in H727 cells as compared with H146 and H345 might warrant further investigation; this discrepancy could indicate altered CFTR processing in carcinoid versus SCLC cell lines, or could be related to the more differentiated state of carcinoid tumors.



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  		Figure 3. Immunoblot analysis and RT-PCR evidence for expression of CFTR in SCLC cell lines H146 and H345 and carcinoid cell line H727. Control tumor cell line T84 expresses CFTR mRNA and not tryptophan hydroxylase mRNA. RT-PCR for actin and {alpha}-tubulin served as reaction and loading controls. Cell extracts were immunopecipitated with anti-CFTR and immunoblotted as described in MATERIALS AND METHODS. NHLF served as control and did not express CFTR protein as expected. Whereas H146 and H345 exhibit a protein banding pattern intensity equivalent to T84, H727 shows a lower CFTR protein expression level relative to the internal actin control. In comparison, H727 shows CFTR mRNA expression level equivalent to or higher than H146 and H345.

 
SCLC Express Functional CFTR with Demonstrable Cl- Secretion
The presence of CFTR mRNA and protein expression in the cells, including membrane localization of CFTR, does not necessarily imply functional Cl- channel activity. To address this issue, we used a functional assay for Cl- secretion based on efflux of preloaded iodide (equivalent ion characteristics to chloride) and detection by an iodide-sensitive electrode. The data shown in Figures 3A–3C demonstrate that iodide loading was as efficient in H146 and H727 cells as in the reference T84 cell line. Treatment of the cells with cAMP agonists to stimulate Cl- conductance showed a burst of iodide efflux within 5 min of incubation in the T84 culture (positive control), as well as the H146 and H727 cultures (Figures 4A–4C). This suggests a common mechanism underlying this iodide efflux. Control cells treated with vehicle only (0.1% DMSO) did not release iodide above baseline values. Similarly, NHLF and the CFTR functionally defective CFPAC-1 cells did not respond to cAMP agonists (Figure 4D).



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  		Figure 4. Iodide efflux assay demonstrating Cl- channel secretory activity in the SCLC cell line H146 and carcinoid cell line H727 as compared with the control tumor cell line T84. As cell controls for the assay, the CFTR nonfunctional cell line CFPAC-1 and nonexpressing NHLF were used. Cells were preloaded with iodide and then stimulated with CFTR agonist cocktail to illicit an iodide secretory response as detected with an iodide-sensitive electrode as described in MATERIALS AND METHODS. Incubation in vehicle alone, 0.1% DMSO, served as an internal control. The cell lines T84 and H727 were transfected with antisense to CFTR, described in MATERIALS AND METHODS, and then assayed for iodide efflux. In A and C, stimulation of H727 and H146 cells with CFTR agonist cocktail produced a significant efflux of iodide within 5 min and ~ 30–50% of that observed in stimulated T84 cells (B). Basal or unstimulated H727, H146, and T84 cells did not secrete iodide (A–C). When H727 and T84 were transfected with antisense to CFTR and then stimulated with agonist cocktail, the iodide efflux response was abrogated, indicating functional inactivation of CFTR and confirming the Cl- conductance measurement. As further validation of the assay, CFTR nonfunctional CFPAC-1 and nonexpressing NHLF cells showed no efflux response to stimulation with agonist cocktail. Iodide equivalent to Cl- concentration was calculated (25) and plotted graphically over time. Values represent triplicates with SEs calculated. Statistical significance was determined with the Mann-Whitney test (significance at P < 0.05).

 
As further evidence that the Cl- secretion observed by efflux assay was due to specific CFTR Cl- channel activity, H727 and T84 cell lines were stably transfected with a CFTR antisense cDNA to downregulate expression of functional CFTR. Cells transfected with antisense CFTR cDNA no longer responded to cAMP agonists, as illustrated by baseline efflux levels only (Figures 4A and 4B).

Effects of CFTR on Stimulus-evoked 5HT Release
The response of H146 to low levels of O2 has been previously described in terms of hypoxia-sensitive ion channels (20). Using a sensitive immunoassay for 5HT, we found that H146 cells respond to hypoxia (5% O2) by rapid secretion of 5HT, ~ 3.7-fold over baseline normoxia level (8.78 ± 1.10 ng 5HT/105 cells versus 2.38 ± 0.49 ng 5HT/105 cells) (Figure 5). High potassium, as expected, also evoked a strong secretion of 5HT (11.19 ± 3.1 ng 5HT/105 cells) within 1 h. In comparison, H146 CFTR--transfected cells showed a markedly reduced (P < 0.0001) secretion of 5HT (2.9 ± 0.81 ng 5HT/105 cells), close to control normoxia levels (2.80 ± 0.78 ng 5HT/105 cells). Similarly, high potassium-stimulated secretion of 5HT (9.13 ± 1.79 ng 5HT/105 cells) from H146 CFTR- cells was significantly reduced (P < 0.01) (Figure 5). As positive and negative controls, H69 secreted 5HT (9.42 ± 1.23 ng 5HT/105 cells) under hypoxic stimulation, whereas no 5HT secretion was detected in T84 cells. A comparable effect, but higher overall level of 5HT secretion, was observed even after 4 h of hypoxia (not shown). These results reveal that downregulation of Cl- conductance significantly inhibits 5HT secretion in SCLC cells, particularly in their acute response to hypoxia.



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  		Figure 5. Secretion of 5HT assessed by ELISA assay showing that 1 h of hypoxia and high K+ concentration (30 mM) induce secretion of 5HT from H146 cells and that this response is inhibited by downregulation of CFTR Cl- conductance in the transfected H146 CFTR- cells. Lack of functional CFTR appears to more specifically affect hypoxia, a physiologic stimulus, as compared with less specific high K+ concentration, although both are significantly different than normoxia-treated cells. In reference, another SCLC cell line, H69, which expresses 5HT responded similarly to hypoxia as did H146, while the 5HT nonexpressing T84 cells were negative. Significance values are indicated by *(P < 0.0001) and **(P < 0.01). Open bar, control; hatched bar, hypoxia; filled bar, high K+.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data demonstrate that SCLC cell lines, H146 and H345, and carcinoid cell line H727, express significant amounts of CFTR mRNA and that the corresponding CFTR protein is localized to both the plasma membrane and cytoplasmic domain of these cells. Using a nonisotopic iodide efflux assay together with an antisense approach for downregulating CFTR expression and function, we also show that CFTR-related Cl- transport is functional in SCLC and carcinoid tumor cell lines. These findings, together with our previous demonstration of functional CFTR in native NEB cells (23), support the notion that CFTR may play an important role in the biology of this neuroendocrine cell type. At present, it is not known whether CFTR demonstrated in different PNEC tumor cell lines is functionally identical to that of normal cells. However, it should be noted that numerous studies in the CF field performed to define the processing, functional activation, and regulation of CFTR are based on experiments using both normal (30) and tumor cell lines, such as T84 (31–33). Based on our findings reported here, SCLC and carcinoid tumor cell lines can serve as potential models to study CFTR-related functions. In addition to previous reports of CFTR expression in CNS neurons (34), we now provide further evidence that CFTR may be functional in peripheral neuroendocrine cell type.

In the context of physiologic functions requiring Cl- transport, the importance of interactions between CFTR and other ion channels and membrane proteins is becoming more appreciated (9, 35, 37). Some examples include interactions with amiloride-sensitive Na+ channel (ENaC) (i.e., when ENac is expressed with CFTR) ENaC activity is altered, as is cAMP stimulation (6) and an interaction between CFTR and the ATP-regulated K+ channel (7). Recent studies suggest that elevated Cl- alone inhibits ENaC activity (36). Plasma membrane Cl- channels are known to play an important role in excitable cells (such as NEB) by stabilizing their membrane potential (35). Since NEB cells possess voltage-activated currents, CFTR Cl- channel functions could potentially interact with these ionic conductances including O2-sensitive K+ channel functions involved in NEB cell O2-sensing mechanism (1, 38). For example, altered CFTR function could result in abnormal membrane signaling (O2 sensing) and/or altered secretory responses (amine/peptide release). As first evidence that CFTR Cl- conductance function may affect neuroendocrine cell functions, we demonstrate that, in H146 cells, downregulation of CFTR Cl- conductance abolishes the acute response to hypoxia and significantly reduces high K+-induced 5HT secretion. Since H146 cells are O2-sensitive, as are native NEB (14, 15, 20), and both express functional CFTR, we suggest the possibility of a close association between CFTR and the O2-sensing mechanism.

We have previously reported expression and localization of CFTR to apical membrane of NEB cells in neonatal rabbits (23). In contrast to normal NEB cells, in SCLC and carcinoid cell lines CFTR is expressed at significantly higher levels and localized to both plasma membrane and cytoplasmic domains similar to that found in T84 tumor cells. This suggests a possible involvement of CFTR in facilitating adaptation of tumor cells to chronic hypoxia and low pH in their microenvironments generated by rapid growth. The reasoning is that, in tumors, hypoxia and acidosis can independently upregulate expression of vascular endothelial growth factor to induce angiogenesis (39, 40). This extracellular acidity, apparently generated via CO2 and lactate, may be linked to activity of Cl- channels because CFTR functions in bicarbonate secretion (41). Therefore, CFTR may play a role in adaptations of tumor cells to adverse microenvironments where low pH can promote invasion and metastasis (42). Thus, CFTR overexpression in pulmonary neuroendocrine tumors may impart biologic properties advantageous for tumor growth and survival. This could also account for a highly malignant behavior and resistance of SCLC to treatment. Although neuropeptide autocrine signaling function in SCLC has been extensively exploited for development of new therapeutic approaches (43), it is conceivable that CFTR might serve as a potential therapeutic target given that downregulating CFTR Cl- conductance affects SCLC response to hypoxia (Figure 5).

Several lines of evidence suggest potential involvement of PNEC/NEB in CF lung disease. Earlier studies reported a significant increase in the frequency of PNEC in lungs of patients who died of CF, thus supporting the possible involvement of these cells in the pathophysiology of CF lung disease (44, 45). A more recent study found increased amounts of bombesin-like peptide in urine from CF patients, suggesting an overactive neuroendocrine cell system (46). Furthermore, bombesin/gastrin-releasing peptide is mitogenic for lung mesenchyme (47, 48), and regeneration of pulmonary epithelium (e.g., Clara cells) appears to focus around NEBs that are located at bifurcations (49). It is therefore possible that in lungs of patients with CF, NEBs, via their secretion of neuropeptides, are essential for maintenance of the bronchopulmonary epithelium either directly or through interaction with adjacent mesenchyme. We have shown previously that receptors for gastrin-releasing peptide can be localized in the surrounding mesenchymal cells and in the submuscosal gland ductal cells in the lung (50). Based on these previous studies, and our recent demonstration of functional CFTR in NEB cells, neuroendocrine functions could play a significant role in the pathogenesis and pathophysiology of CF lung disease (23). Because PNEC/NEB cells are an integral component of airway mucociliary epithelium, the potential mechanisms in PNEC/NEB of patients with CF (and having deficient CFTR function) include abnormal membrane signaling and/or altered secretion of amine and peptide mediators. This in turn could contribute to mucus hypersecretion, altered periciliary fluid, and local chemotaxis of inflammatory cells (51–55). The latter may be particularly relevant to the early pre-infectious stage of CF lung disease when PNEC/NEB are most prominent (2, 53). Finally, based on the data presented here, it is conceivable that, in perinatal lungs of CF patients lacking functional CFTR, altered neuroendocrine cell response to hypoxia could lead to aberrant secretion of amine and/or peptides, thereby affecting lung physiology. PNEC/NEB-relevant CFTR-expressing tumor models characterized in this study may provide a readily accessible resource for investigating the neuroendocrine/CFTR relationship under normal and various pathologic conditions.


   Acknowledgments
 
The authors thank Dr. A. Pavirani (Transgene, Strasbourg, France) for the gift of the anti-CFTR antibodies (MATG1061 and MATG1031). They also thank Dr. Canhui Li for assistance in development of the iodide efflux assay and Soma Choudhury for generation of the CFTR antisense construct. The work was funded in part by grants received from the Canadian Cystic Fibrosis Foundation and Canadian Institutes of Health Research to E.C., H.Y., and C.B.

Received in original form February 19, 2002

Received in final form May 31, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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