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
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ABSTRACT |
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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
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INTRODUCTION |
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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., 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.
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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.
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
· 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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RESULTS |
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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.
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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
· 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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DISCUSSION |
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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 -interferon, thereby
promoting cell proliferation. The ability to culture the MTE18-(
/
)
and the MTE7b-(+/
) cell lines at 33°C in the absence of
-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
-interferon were stained with the anti-TAg antibody. The
phenotype-dependent nature of the requirement of cells from
H-2Kb-tsA58 mice for
-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
-interferon is required for growth.
Interestingly, when attempts were made to culture either of the MTE
cell lines in the presence of
-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
· 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.
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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.
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