Expression of nucleotide-regulated Clminus currents in CF and normal mouse tracheal epithelial cell lines

E. J. Thomas*,1, S. E. Gabriel*,2, M. Makhlina2, S. P. Hardy1, and M. I. Lethem1

1 School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, United Kingdom; and 2 School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The dominant route for Cl- secretion in mouse tracheal epithelium is via Cl- channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(-/-)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/-)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10-4 M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current (Isc) and resistance of both cell lines, with larger increases in Isc being elicited by ionomycin (10-6 M). Both cell lines expressed P2Y2 receptors and responded to the purinergic agonists ATP, UTP, and 5'-adenylylimidodiphosphate (10-4 M) with an increase in Isc. This response could be inhibited by DIDS and was abolished in the presence of Cl--free Ringer solution. Reducing the mucosal Cl- concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(-/-) cells. Pretreatment of these cells with thapsigargin caused a direct increase in Isc and inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl- currents and may prove valuable tools in studying the properties of this pathway.

cystic fibrosis transmembrane conductance regulator; T antigen; Immortomouse; conditionally immortal; purinergic


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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CYSTIC FIBROSIS (CF) is an autosomal recessive disease resulting from mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), which is widely expressed in epithelial tissues and acts as a cAMP-regulated Cl- channel (1-3, 24). In addition, CFTR has been shown to regulate the activity of other proteins, in particular the amiloride-sensitive epithelial Na+ channel (38). The loss of functional CFTR in CF epithelia thus results in altered ion and fluid transport and the production of dehydrated epithelial mucus secretions, which obstruct the lumens of glands and ducts and cause the characteristic manifestations of CF. In humans, several organ systems are involved, including the pancreas and gastrointestinal tract but, most prominently, the respiratory tract, which in CF is characterized by the hypersecretion of highly viscid mucus and chronic bacterial infection.

More recently, several transgenic mouse models of CF have been generated incorporating a knockout of CFTR or one of the CFTR mutations more commonly found in human populations (i.e., Delta F508 or G551D) (12, 14, 15, 29, 33, 37, 39). However, the value of these mice as models of CF has been limited, because they do not display the pulmonary manifestations that characterize CF in humans, although they do exhibit a gastrointestinal phenotype that is generally similar to that of humans (20). This difference in pathology is probably due to the fact that, unlike human airways, the dominant Cl- conductance of mouse trachea is via a Ca2+-activated Cl- channel (CaCC), rather than CFTR (21), and the activity of this alternative Cl- conductance protects the airways from the loss of CFTR (10). The presence of an equivalent to CaCC in human airway epithelia has been established in normal and CF tissues (4); however, in this case, its unstimulated activity is too low to permit a protective role in these tissues. Nevertheless, this Ca2+-activated pathway can be stimulated in CF tissues, and it has been suggested that activation of CaCC by purinergic agonists may have a beneficial effect on airway disease in CF (10). Despite the therapeutic potential of this approach, the properties of CaCC are not well characterized, in part because of the lack of a suitable model system in which to conduct such studies. The purpose of this study was to generate novel tracheal epithelial cell lines from CF and non-CF transgenic mice, thereby providing model systems in which the ion transport properties of mouse tracheal epithelium, and in particular those of CaCC, could be explored further.


    MATERIALS AND METHODS
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Animal husbandry. Mice possessing the simian virus 40 (SV40) large T antigen (TAg) oncogene and the various CFTR genotypes were bred. Heterozygous cftrtm1Unc mice (Jackson Laboratory, Bar Harbor, ME) were crossed with hemizygous H-2Kb-tsA58 mice (Immortomouse, Charles River, Margate, Kent, UK), which possess the gene for the tsA58 temperature-sensitive form of TAg (23). Offspring from this cross were genotyped with respect to CFTR and TAg, and those mice that were heterozygous for the CFTR knockout and the TAg were interbred to produce mice with the various possible combinations of genotype with respect to CFTR and TAg. Mice that were heterozygous for the CFTR knockout and possessed the TAg were interbred to maintain the mouse colony; those with TAg and the various CFTR genotypes, homozygous knockout [CFTR-(-/-)], heterozygous [CFTR-(+/-)], and wild type [CFTR-(+/+)], were used for the initiation of cell lines. To reduce the high mortality rates due to intestinal obstruction exhibited by the cftrtm1Unc mice, all mice were provided with an electrolyte solution containing 6% polyethylene glycol (Colyte) instead of drinking water (19). As a result, the life expectancy of cftrtm1Unc mice was similar to that of wild-type mice and was limited only by the thymic hyperplasia characteristic of H-2Kb-tsA58 mice.

Genotyping of mice and cell lines. The CFTR and TAg genotype of mice and cell lines was determined by PCR of genomic DNA isolated from tail tip or cell cultures with use of commercial kits for DNA isolation (Puregene, Flowgen Instruments, Lichfield, UK) and PCR (Dynazyme, Flowgen Instruments). PCR of TAg was performed using a sense primer sequence of 5'-AAGCTTGTAACAGAGTATGCAATG-3' and an antisense primer sequence of 5'-AAGCTTTCCCCCCACATAATTC-3' and commenced with a denaturation period of 5 min at 95°C, after which Taq polymerase (1 U) was added to each reaction, and 25 cycles of a program of 94°C (1 min), 60°C (1 min), and 70°C (4 min) were run, giving a 532-bp TAg product. PCR of CFTR was performed using the primers CFM1 (5'-TCACTCCTGATGTTGATTTTGGGAGA-3'), CFM2 (5'-ACCTGCTGTAGTTGGCAAGCTTTGAC-3'), and CFM3 (5'-GACGCTGGGCGGGGTTTGCTCGACA-3') and commenced with a denaturation period of 5 min at 95°C, after which Taq polymerase (1 U) was added to each reaction, and 35 cycles of a program of 93.5°C (1 min 15 s), 58°C (1 min 15 s), and 72°C (2 min 15 s) were run. By use of this protocol, the CFTR knockout (S489X) gave a 210-bp product and the wild-type CFTR yielded a 190-bp product. PCR products were identified by electrophoresis on 2% (wt/vol) and 3% (wt/vol) agarose gels for TAg and CFTR products, respectively.

RT-PCR of P2Y2 receptors. Total RNA was isolated from MTE18-(-/-) and MTE7b-(+/-) cells grown on permeable supports by conventional organic phase-separation techniques (7). RT of 1 mg of total RNA was carried out using random hexamer priming at 50°C for 60 min with ThermoScript reverse transcriptase (GIBCO BRL), and 5% of the RT reaction was used as a template for the PCR. Primers for the P2Y2 receptor message were 5'-TTCCGATCACTTGACCTCAGCT-3' (sense) and 5'-AATGTCCTTAGTCTCACTTCCA-3' (antisense), giving a 302-bp product. Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5'-AATGTGTCCGTCGTGGATCT-3' (sense) and 5'-CCCTGTTGCTGTAGCCGTAT-3' (antisense), giving a 255-bp product (accession number M32599). PCR was performed under the following conditions: initial denaturation at 94°C for 3 min and 40 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 45 s. GAPDH amplification was carried out for a total of 30 cycles.

Establishment and culture of cell lines. Two tracheal cell lines were established from mice that possessed the TAg: one from a mouse homozygous for a CFTR knockout [designated MTE18-(-/-)] and the other from a mouse heterozygous with respect to the CFTR knockout [designated MTE7b-(+/-)]. To initiate cultures, tracheae were removed from mice (5-10 wk of age), cleaned of connective tissue in a dish of MEM (Joklik's modification, Life Technologies, Paisley, UK), and cut into rings. The tracheal rings were opened out into arcs by cutting through the posterior membrane, and three to four arcs were placed on their side on a premoistened 12-mm culture insert (Transwell-col, Corning-Costar, High Wycombe, UK). Culture medium, a 1:1 mixture of Ham's F-12 and 3T3 fibroblast-conditioned medium (13) supplemented with transferrin (2.5 µg/ml), insulin (5 µg/ml), epidermal growth factor (12.5 ng/ml), endothelial cell growth supplement (1.875 µg/ml), triiodothyronine (15 nM), hydrocortisone (0.5 µM), and CaCl2 (0.5 mM), was placed beneath the insert and a few drops on the tracheal rings, and the explants were cultured at 37°C until the cells had grown to confluence. Then the cells were passaged and expanded onto fresh inserts and cultured at 33°C, the permissive temperature for TAg activity. Once established, both cell lines were maintained as bulk cultures, as opposed to clonal populations.

Characterization of cell lines. The epithelial nature of MTE7b-(+/-) and MTE18-(-/-) cell lines was characterized immunohistochemically using antibodies to epithelium-specific cytokeratins and the fibroblast protein vimentin. For immunohistochemistry, cells were cultured on glass coverslips for 72 h at 33°C, fixed in 1:1 ethanol-acetone for 3 min at room temperature, and washed in PBS. Fixed cells were incubated at 4°C overnight with primary antibody [anti-pan cytokeratin antibody or anti-vimentin antibody (Sigma Chemical) diluted 1:60 and 1:30, respectively, in PBS containing 1% (vol/vol) FCS]. After incubation with primary antibody, cells were washed repeatedly in PBS and then in distilled H2O and incubated at 4°C overnight with FITC-conjugated rabbit anti-mouse Ig secondary antibody (Dako, Cambridge, UK) diluted 1:100 in PBS containing 1% (vol/vol) FCS. Cells were mounted using Vectashield fluorescence mounting medium (Vector Laboratories, Peterborough, UK) and visualized by fluorescence microscopy. To confirm the TAg-positive nature of the cell lines, the immunofluorescence procedure was repeated using an anti-TAg antibody (clone OH-1, Biogenesis, Poole, UK) diluted 1:100 in PBS containing 1% (vol/vol) FCS. Appropriate reagent controls were included in all experiments, and 3T3 mouse fibroblasts and MGEN mouse gallbladder epithelial cells (a gift from Dr. L. Clarke, University of Missouri) were used as cell controls.

The temperature-sensitive nature of the immortalization of MTE7b-(+/-) and MTE18-(-/-) cell lines was investigated by comparing the colony-forming efficiency of the cells cultured at 33°C, the permissive temperature for TAg activity, with that of the cells at 39.5°C, the nonpermissive temperature for TAg activity. Cells were seeded at clonal density (103 cells/60-mm dish) and cultured at 33°C for 24 h, and then half of the cultures were transferred to 39.5°C and cultured for a further 10 days. Cultures were fixed and stained for 5 min with 50% (vol/vol) ethanol containing 2% (wt/vol) methylene blue, rinsed with water, and air dried, and the colonies were counted. Colony-forming efficiency was calculated as the number of colonies formed expressed as a percentage of the total number of cells seeded.

Bioelectric measurements. Bioelectric measurements were conducted using Ussing chambers on cells cultured at 33°C and between passages 42 and 80 and between passages 50 and 74 for MTE18-(-/-) and MTE7b-(+/-) cells, respectively. Cells were cultured on collagen matrix supports and studied as described previously (41) or cultured on culture inserts (Falcon, Becton Dickinson, Bedford, MA) coated with type 1 rat tail collagen (Sigma Chemical). To reduce edge damage, cells grown on inserts were plated into the center of rings of silicone elastomer (Sylgard 184, Dow Corning) that were attached to the insert membrane by rubber glue. All cells were cultured at 33°C and studied at 3-4 days after confluence when the membrane resistance was >150 Omega  · cm2. Electrical measurements were made under short-circuit conditions in conventional Ussing chambers with the voltage of the mucosal bathing solution clamped to zero and transepithelial resistance (RT) calculated according to Ohm's law from the magnitude of current deflections in response to voltage pulses of 1 mV for 0.35 ms every 25 s.

Solutions and drugs. Once mounted in Ussing chambers, cells were bathed on both sides with Krebs-Ringer bicarbonate solution, pH 7.4, containing (in mM) 113 NaCl, 4.5 KCl, 1.2 Na2HPO4, 25 NaHCO3, 10 glucose, 1.1 CaCl2, and 1.2 MgCl2, which was warmed to 37°C and gassed with 95% O2-5% CO2, except in experiments to study the role of Cl- in bioelectric responses where NaCl in the Ringer solution was substituted with sodium gluconate. In some experiments the solution bathing the mucosal surface of the cells was altered to a low-Cl--high-K+ Ringer solution of the following composition (in mM): 147 K+, 40 Na+, 4.8 Cl-, 1.2 Mg2+, and 1.2 Ca2+, thereby increasing the driving force for Cl- transport while eliminating any effects due to the activation of apical K+ channels. All drugs were applied to the mucosal surface of the cells, with the exception of ionomycin and thapsigargin, which were added to the serosal and mucosal bathing solutions. Amiloride, dibutyryl cAMP, DIDS, forskolin, IBMX, and ionomycin were obtained from Sigma Chemical. Adenylyl imidodiphosphate tetralithium salt (AMP-PNP), ATP, and UTP were obtained from Boehringer Mannheim (Indianapolis IN or Lewes, UK). Thapsigargin was obtained from Molecular Probes (Eugene, OR).


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MATERIALS AND METHODS
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Generation and characterization of cell lines. In the present study it proved possible to breed mice possessing the TAg gene and the various possible CFTR genotypes. Because of the inability of PCR to distinguish between animals that were hemizygous and homozygous for TAg, the full genotypes of the animals were not known, and expected percentages of particular genotypes could only be calculated as a range. The genotypes of the animals in generations F3-F6 were as follows: 20.3% (range 18.75-25%) CFTR-(+/+) TAg-(+), 12.7% (range 0-6.25%) CFTR-(+/+) TAg-(-), 44.9% (range 37.5-50%) CFTR-(+/-) TAg-(+), 14.4% (range 0-12.5%) CFTR-(+/-) TAg-(-), 5.1% (range 18.75-25%) CFTR-(-/-) TAg-(+), and 2.5% (range 0-6.25%) CFTR-(-/-) TAg-(-), a pattern that generally follows that predicted by Mendelian laws.

Both cell lines established from this colony of mice, MTE18-(-/-) and MTE7b-(+/-), grow with a cobblestone appearance characteristic of many epithelia and, when grown on inserts, form monolayers of generally cuboidal morphology (Fig. 1, A and B). The established cell lines exhibit similar proliferation rates (population doubling time of 1.5 days) and have been cultured to >400 population doublings without any change in their morphological or bioelectric characteristics. However, during the early stages of expansion, both cell lines underwent periods in which extensive cell death was evident and cell growth slowed to a point at which the cell number in the cultures remained stationary. The cell lines appeared to be at risk of these periods of reduced proliferation until they had undergone 30-40 population doublings, then proliferation rates stabilized.


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Fig. 1.   Characterization of MTE18-(-/-) and MTE7b-(+/-) cell lines. A and B: hematoxylin-and-eosin-stained sections of MTE18-(-/-) and MTE7b-(+/-), respectively, cultured on tissue culture inserts at 33°C for 5 days (magnification ×250). C: immunohistochemical staining of MTE18-(-/-) cells with anti-pan cytokeratin antibody [magnification ×650; similar results were obtained with MTE7b-(+/-) cells, results not shown]. D: immunohistochemical staining of MTE7b-(+/-) cells with anti-T antigen (TAg) antibody [magnification ×650; similar result were obtained with MTE18-(-/-) cells, results not shown]. E and F: genotype analysis of MTE cells by PCR of cystic fibrosis transmembrane conductance regulator (CFTR) gene [lane 1, MTE18-(-/-); lane 2, MTE7b-(+/-); lane 3, DNA ladder] and TAg gene (lane identities as for E), respectively.

The epithelial nature of the cell lines was verified by immunofluorescence staining with anti-pan cytokeratin and vimentin antibodies, which indicated expression of epithelial cytokeratins (Fig. 1C) but not the fibroblast-specific protein vimentin (results not shown). The CFTR and TAg genotype of each cell line was confirmed by PCR, which indicated that MTE18-(-/-) cells were homozygous for the S489X CFTR knockout, MTE7b-(+/-) cells were heterozygous for this mutation (Fig. 1E), and both cell lines were positive for TAg (Fig. 1F). Both cell lines also gave positive nuclear staining with the anti-TAg antibody, indicating that TAg protein is expressed (Fig. 1D). Culture of the cell lines at the nonpermissive temperature of 39.5°C reduced the colony-forming efficiency to 14.1% [MTE18-(-/-)] and 36.5% [MTE7b-(+/-)] of that seen when the cells were cultured at the TAg permissive temperature of 33°C (Fig. 2), thereby indicating the conditional nature of the immortalizations. This aspect was indicated further by the fact that it was not possible to maintain cultures of either cell line at the nonpermissive temperature (39.5°C).


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Fig. 2.   Conditional immortalization of MTE cells shown as colony-forming efficiencies of MTE18-(-/-) and MTE7b-(+/-) cells cultured at the permissive temperature (33°C) and the nonpermissive temperature (39.5°C). Values are means ± SE from >= 10 experiments. ***P < 0.001 (Mann-Whitney test).

Bioelectric studies. When grown on tissue culture inserts, both cell lines formed polarized monolayers that, when mounted in Ussing chambers, generated transepithelial potential differences (PD) and short-circuit currents (Isc) with moderate RT (Table 1). Basal PD and RT were significantly greater in the MTE7b-(+/-) cell line, whereas the Isc of the MTE18-(-/-) cells was significantly greater than that of the heterozygous cells. Culture of both cell lines at 37.5°C, a condition designed to maximize differentiation of the cells by permitting only low levels of TAg activity, had no effect on basal bioelectric properties (results not shown). Addition of amiloride to the mucosal bathing solution resulted in a small but not significant drop in the Isc of both cell lines (Table 1), indicating that Na+ conductance is not a significant component of the basal Isc. Stimulation of cAMP-dependent pathways by forskolin or IBMX had no effect on the Isc of either cell line, a lack of effect that was maintained even under maximal stimulation of these pathways by a cocktail of forskolin, IBMX, and dibutyryl cAMP (Table 1). Stimulation with the cAMP cocktail did, however, result in small increases in the RT of 18.05 ± 4.55 and 24.67 ± 8.67 Omega  · cm2 for MTE18-(-/-) and MTE7b-(+/-) cells, respectively. Addition of the Ca2+ ionophore ionomycin to mucosal and serosal bathing solutions induced a significant increase in the Isc of both cell lines (Table 1), indicating the presence of Ca2+-activated currents.

                              
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Table 1.   Bioelectric properties of MTE18-(-/-) and MTE7b-(+/-) cell lines

In both cell lines, the mucosal addition of the purinergic agonist ATP caused a large increase in the Isc (Fig. 3, A and B) that returned slowly to near baseline levels over ~30 min (data not shown). In both cases, the increase in Isc could be significantly reduced by the subsequent addition of the Cl- channel blocker DIDS (Fig. 3, A and B). The ability of DIDS to inhibit the ATP-stimulated change in Isc was further investigated by pretreatment of the cells with DIDS for 10 min before the addition of ATP. DIDS pretreatment attenuated the change in Isc induced by ATP in both cell lines, although the extent of attenuation was significantly greater in the MTE18-(-/-) cells than in the MTE7b-(+/-) cells (96.2 ± 1.0 vs. 56.3 ± 4.3%, P < 0.01; data not shown). Changes in Isc similar to those observed after the addition of ATP were also seen when the cells were treated with the ATP analog AMP-PNP or the P2Y2 receptor agonist UTP (Fig. 3C). In experiments where Cl- in the bathing medium was replaced with gluconate, the response of MTE18-(-/-) cells to mucosal UTP (10-4 M) was almost entirely abolished, the change in Isc being reduced to 2.5 ± 1.2 (SE) µA/cm2 (n = 7, P < 0.001, t-test). In addition, both cell lines were shown to express P2Y2 receptors by RT-PCR, as seen by the 302-bp product in both cell lines (Fig. 3D).


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Fig. 3.   Nucleotide-regulated changes in the short-circuit current (Isc) of MTE cells and expression of P2Y2 receptors by MTE cells. A and B: recorder tracings of MTE18-(-/-) and MTE7b-(+/-) cells, respectively, exposed to mucosal ATP (10-4 M) and then bilateral DIDS (10-4 M). C: peak Isc responses of MTE cells to ATP (solid bars), UTP (open bars), and AMP-PNP (hatched bars). All nucleotides were 10-4 M in the mucosal bath. Values are means ± SE from >= 5 experiments. P < 0.01 for all responses except ATP and UTP on MTE18-(-/-), where P < 0.001 (Mann-Whitney test). D: RT-PCR amplification of P2Y2 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message in MTE cells [lanes 1 and 2, P2Y2 in MTE7b-(+/-) and MTE18-(-/-) cells, respectively; lane 3, DNA ladder; lanes 4 and 5, GAPDH in MTE7b-(+/-) and MTE18-(-/-) cells, respectively].

The bioelectric response to UTP could be increased in both cell lines by replacing the mucosal solution with a high-K+-low-Cl- Ringer solution, thereby increasing the driving force for the movement of Cl- from the serosal to the mucosal bath (Fig. 4, A and B). The UTP-stimulated Isc was reduced by the subsequent addition of DIDS and stabilized at a level slightly above that of the basal current (10-15% of the peak current induced by UTP; Fig. 4, A and B). The size of the response to UTP under conditions of increased driving force was considerably greater in MTE18-(-/-) than in MTE7b-(+/-) cells. Studies of the dose-response relationship to UTP (Fig. 4C) indicated that this difference between the cell lines was due primarily to the fact that the maximum response of MTE18-(-/-) cells was ~2.5 times that of the MTE7b-(+/-) cells, rather than a change in the sensitivity of the MTE18-(-/-) cells to UTP [EC50 = 5.1 × 10-7 and 3.6 × 10-7 for MTE18-(-/-) and MTE7b-(+/-) cells, respectively].


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Fig. 4.   Stimulation of Isc by UTP in MTE cells bathed with mucosal high-K+-low-Cl- Ringer solution. A and B: recorder tracings of MTE18-(-/-) and MTE7b-(+/-) cells, respectively, bathed mucosally in high-K+-low-Cl- Ringer solution and exposed to mucosal UTP (10-4 M) and then to bilateral DIDS (5 × 10-4 M); inset: tracing from B with expanded scale. C: dose-response curves for the effect of UTP on Isc in MTE18-(-/-) cells () and MTE7b-(+/-) cells () exposed to mucosal high-K+-low-Cl- Ringer solution. Values are means ± SE from >= 5 determinations.

Treatment of the MTE18-(-/-) cells with thapsigargin (10 nM), an inhibitor of the Ca2+-ATPase responsible for refilling intracellular Ca2+ stores, resulted in a sustained increase in Isc and an attenuation of the Isc response elicited by subsequent addition of UTP (Fig. 5A). Similar effects of thapsigargin on Isc and the response to subsequent UTP treatment were also observed in the MTE7b-(+/-) cells (results not shown). The response of MTE18-(-/-) cells to thapsigargin was dose dependent in the range 0-1 µM, with the highest doses resulting in an increase in Isc of 28.2 ± 5.0 µA/cm2 (Fig. 5B). The response of the cells to a subsequent challenge with UTP (10-4 M) was inhibited by thapsigargin in a dose-dependent fashion (Fig. 5B). Doses of thapsigargin as low as 3 nM caused a 25% reduction in the response to the UTP challenge, with further reductions in the UTP response being seen in cells treated with higher doses of thapsigargin and 1 µM thapsigargin causing a maximum inhibition of 96%.


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Fig. 5.   Effect of thapsigargin on basal and UTP-stimulated Isc in MTE18-(-/-) cells bathed with mucosal high-K+-low-Cl- Ringer solution. A: recorder tracing of MTE18-(-/-) cells exposed to mucosal thapsigargin (Thapsi, 10-8 M) and then to UTP (10-5 M). B: effect of various doses of mucosal thapsigargin (hatched bars) on the Isc of MTE18-(-/-) cells and the response to subsequent mucosal UTP (10-5 M; solid bars). Values are means ± SE; n = 8.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The mouse tracheal epithelium, despite expressing little or no CFTR (21, 43), represents a useful model for the study of alternative pathways for Cl- secretion, which may be of therapeutic value in the treatment of CF. One limitation of murine models, however, is the small size of the tissues, such that up to four mice are required to produce a single culture for Ussing chamber studies (9). Production of the mouse tracheal epithelial cell lines that maintain the ion transport characteristics of native epithelium should alleviate this problem. In the present study, two such cell lines have been generated: one from a mouse homozygous for the S489X CFTR knockout and one from a mouse heterozygous for this mutation. The generation of these cell lines by crossing CF mice with mice possessing the TAg transgene demonstrates that this is a simple method for introducing the immortalizing TAg gene into mouse cells from other transgenic strains and thereby isolating cell lines of interest. One potential limitation of this approach is the possibility that the gene of interest might be in close proximity to the site of TAg on the mouse genome. However, in the present study the finding that the pattern of inheritance broadly followed Mendelian laws indicates that the TAg and CFTR genes are sufficiently separated on the genome.

Defined hormone-supplemented low-serum media inhibit fibroblast growth, thereby promoting the selection of other cell types in a culture. The use of such a medium in the present study has aided the isolation of cell lines that are epithelial in nature, as indicated by their positive cytokeratin staining and negative vimentin staining. The use of the H-2Kb promoter in the Immortomouse is designed to permit upregulation of TAg expression by gamma -interferon, thereby promoting cell proliferation. The ability to culture the MTE18-(-/-) and the MTE7b-(+/-) cell lines at 33°C in the absence of gamma -interferon raises the possibility that these immortalizations may not have occurred in a TAg-dependent manner. However, the conditional nature of the immortalizations, as indicated by the reduction in colony-forming efficiency at 39.5°C and the inability to culture the cell lines at this temperature, indicates that active TAg is required by these cell lines. It is likely, therefore, that the basal activity of the H-2Kb class 1 promoter in tracheal epithelium is sufficient to drive TAg expression, a suggestion that is supported by the strong signal obtained when cells grown in the absence of gamma -interferon were stained with the anti-TAg antibody. The phenotype-dependent nature of the requirement of cells from H-2Kb-tsA58 mice for gamma -interferon has been noted previously (23, 28, 40) and supports further the notion that it is the endogenous promoter activity of these cells that determines whether gamma -interferon is required for growth. Interestingly, when attempts were made to culture either of the MTE cell lines in the presence of gamma -interferon, a reduction in cell proliferation and increased cell detachment was observed. The growth-inhibitory activity of interferons has been reported previously in other cell types (18, 32, 35) and, in this case, was obviously strong enough to overcome the proliferative drive resulting from TAg expression.

The MTE cell lines in this study exhibited basal RT values that were broadly similar to those that have been reported for primary cultures of airway epithelium from several species including mouse (200-420 Omega  · cm2) (5, 9, 41). However, the basal PD and Isc of the cell lines were less than those found in primary mouse tracheal epithelial cultures (9). One possibility is that this reduction in basal activity is the result of the highly proliferative nature of the cell lines when grown at 33°C with a consequent loss of differentiation. However, the relatively high RT values of the cell lines and their cuboidal morphology in culture suggest that they retain reasonable levels of differentiation when cultured at 33°C. Furthermore, the basal bioelectric parameters of the cells were unchanged when the cells were cultured at 37.5°C to reduce TAg activity, an approach that increases the levels of differentiation of rat intestinal epithelial cells immortalized with the tsA58 TAg (31).

Several studies have indicated that, compared with human epithelium, cultured and freshly excised mouse tracheal epithelium has a reduced amiloride-sensitive Na+ current (9, 21, 33, 36). In the present study, addition of amiloride to the mucosal bathing solution had no significant effect on the Isc of either cell line, suggesting little or no basal Na+ conductance in the apical membrane of these cells. The small contribution of this current to the bioelectric properties of the cell lines may help explain their unusually low basal Isc values.

CFTR is known to act as a cAMP-regulated Cl- channel, and the loss of the resulting cAMP-activated current in tissues that express CFTR, e.g., human airway and mouse nasal epithelia, is a distinguishing feature of CF (4, 11, 22). In the present study, neither the MTE18-(-/-) nor the MTE7b-(+/-) cells responded to a cocktail of drugs known to activate intracellular cAMP-dependent pathways. Activation of these pathways in freshly excised mouse tracheal tissues has been associated with a Cl- secretory response (11, 21, 36) that does not appear to be mediated by CFTR, since it is present in CFTR knockout mice (21) and mouse trachea expresses little or no CFTR (43). Grubb et al. (21) showed that treatment of these tissues with forskolin results in an increase in intracellular Ca2+ concentration ([Ca2+]i), raising the possibility that the Cl- secretory response to cAMP is mediated via the CaCC, which is the dominant Cl- secretory pathway in mouse trachea (21). Interestingly, this cross talk between the cAMP- and Ca2+-dependent pathways was present in freshly excised tissues but not in primary cultured mouse tracheal cells. Hence, the lack of response to cAMP of the MTE18-(-/-) and MTE7b-(+/-) cell lines remains consistent with this hypothesis.

In view of the dominance of the Ca2+-activated pathway for Cl- transport in mouse trachea, the cell lines were treated with the Ca2+ ionophore ionomycin to confirm the presence of such a pathway. MTE18-(-/-) and MTE7b-(+/-) cell lines responded similarly to ionomycin with highly significant increases in Isc that were accompanied by significant increases in PD. The magnitude of the response to ionomycin in the MTE cell lines was ~50% of that reported for primary cultures of mouse airway epithelium (10); however, this is probably due to the higher concentration of ionomycin used in the previous study than used here.

Previous studies have indicated that in airway epithelia the Ca2+-activated Cl- secretory pathway can be stimulated by the action of purinergic agonists on P2Y2 receptors (27). MTE18-(-/-) and MTE7b-(+/-) cell lines responded to the mucosal addition of the purinergic agonists ATP, AMP-PNP, and UTP with a large increase in Isc, suggesting that these cells have retained this pathway. This possibility is strengthened further by the ability of the poorly hydrolyzable ATP analog AMP-PNP to elicit a response similar to that seen with ATP, indicating that ATP is acting directly, rather than as a breakdown product, whereas the equipotency of UTP and ATP in these cells is consistent with the known agonist profile of P2Y2 receptors (16, 34). In addition, the expression of P2Y2 receptors in both cell lines was confirmed by RT-PCR. Interestingly, the response of both cell lines to nucleotides was greater than the response to ionomycin (Table 1). Although both these stimuli are known to increase intracellular Ca2+, it is possible that nucleotides are also acting via some additional mechanism and thereby generating a greater increase in Isc. However, the possibility that both are acting solely via intracellular Ca2+ and that the different responses are due to differences in the characteristics of the Ca2+ increase elicited by the two agents e.g., via different local pools of Ca2+ within the cell, cannot be ruled out.

Several lines of evidence suggest that the identity of the purinergic-activated conductance in the cell lines is Cl-, including the ability of DIDS to substantially inhibit the response to ATP. In addition to its activity as a Cl- channel blocker, DIDS has been reported to act as an antagonist at P2X receptors (8); however, there is no evidence for such an action at P2Y2 receptors, and therefore it seems likely that it is the channel-blocking mechanism that is responsible for the action of DIDS in this study. Interestingly, the DIDS-inhibitable current was significantly greater in MTE18-(-/-) than in MTE7b-(+/-) cells, raising the possibility that ATP may be stimulating currents other than Cl- in the heterozygous cells. However, in MTE18-(-/-) cells, the identity of the ATP-stimulated current as Cl- was confirmed by the fact that the response to UTP could be almost entirely eliminated when Cl- was substituted with the nontransported anion gluconate in the bathing medium.

In line with the purinergic-activated conductance being Cl-, the response to UTP in both cell lines was enhanced when the driving force for Cl- secretion was increased by changing the mucosal bathing medium to low-Cl- Ringer solution. Furthermore, this conductance could be substantially inhibited by DIDS. In the presence of this increased driving force for Cl- secretion, the maximal response to UTP was considerably greater in the MTE18-(-/-) than in the MTE7b-(+/-) cells (Fig. 4). The most likely explanation for this difference is that the MTE18-(-/-) cells express a greater number of CaCC, which only becomes apparent in the presence of powerful stimulus for Cl- secretion through this pathway (i.e., low mucosal Cl-).

The contention that the purinergic-activated Cl- current in the MTE cell lines is mediated via the mobilization of Ca2+ was investigated using the Ca2+-ATPase inhibitor thapsigargin, which has been shown to increase [Ca2+]i and deplete intracellular Ca2+ stores (6). Thapsigargin induced a dose-dependent increase in the Isc of MTE18-(-/-) cells bathed mucosally with high-K+-low-Cl- Ringer solution. The ability of thapsigargin to increase [Ca2+]i in several airway epithelia has been reported previously (26, 30, 42), and the results obtained here are consistent with a rise in [Ca2+]i causing activation of CaCC.

In addition to its ability to increase Isc, thapsigargin also inhibited the response of the MTE18-(-/-) cells to the subsequent application of UTP. The fact that the combined increase in Isc due to thapsigargin and UTP decreased by nearly fourfold as the thapsigargin dose rose from 0 to 1,000 nM indicates that this effect is not merely due to the cells responding maximally under the combined influence of thapsigargin and UTP but represents a true inhibitory action of thapsigargin. This finding is consistent with previous studies in which thapsigargin was shown to block the ATP-stimulated increase in [Ca2+]i of airway epithelia (17, 26), whereas the inhibitory activity of thapsigargin on Ca2+-mediated purinergic responses in airway epithelia was noted in another study (25). As a result, the probable explanation for the action of thapsigargin in MTE18-(-/-) cells is a direct activation of Ca2+-activated conductances and depletion of intracellular Ca2+ stores, leading to an inhibition of Ca2+-mediated Cl- secretion in response to UTP. These actions of thapsigargin are entirely consistent with the expression of CaCC in MTE18-(-/-) cells and its activation by UTP.

In summary, two mouse tracheal epithelial cell lines, one CF and the other heterozygous for CFTR, have been generated. Unlike primary cultures of mouse tracheal epithelium and freshly excised tissues, the cell lines do not express a basal Na+ conductance, but in other respects the pattern of transport resembles that reported for these other systems. In particular, the cell lines express P2Y2 receptors and retain an intact purinergic-regulated Cl- secretory pathway that is mediated via changes in [Ca2+]i and probably represents the alternative Cl- channel, CaCC, which has been described in mouse and human airway epithelia. As a result, these cell lines represent a plentiful and useful model for studies of the properties of CaCC. In view of the suggestion that activation of CaCC in airway epithelia may be beneficial in CF, these studies may well prove valuable in defining the potential role of CaCC in the treatment of CF.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Elena Martsen for molecular biology expertise and guidance with RT-PCR.


    FOOTNOTES

* E. J. Thomas and S. E. Gabriel contributed equally to this work.

This work was supported by Wellcome Trust Grants 041480/Z/94/Z (M. I. Lethem and E. J. Thomas) and 047407/Z/96/Z (S. P. Hardy) and Cystic Fibrosis Foundation Grant Gabrie96P0 (S. E. Gabriel and M. Makhlina).

Address for reprint requests and other correspondence: M. I. Lethem, School of Pharmacy and Biomolecular Sciences, University of Brighton, Lewes Rd., Brighton BN2 4GJ, UK (E-mail: m.i.lethem{at}brighton.ac.uk).

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 4 November 1999; accepted in final form 25 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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