Am J Physiol Cell Physiol
279: C1578–C1586, 2000.
Expression of nucleotide-regulated Cl⫺ currents
in CF and normal mouse tracheal epithelial cell lines
E. J. THOMAS*,1 S. E. GABRIEL*,2 M. MAKHLINA,2 S. P. HARDY,1 AND M. I. LETHEM1
School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ,
United Kingdom; and 2School of Medicine, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599
1
Received 4 November 1999; accepted in final form 25 May 2000
cystic fibrosis transmembrane conductance regulator; T antigen; Immortomouse; conditionally immortal; purinergic
(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 epitheCYSTIC FIBROSIS
* E. J. Thomas and S. E. Gabriel contributed equally to this work.
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
@brighton.ac.uk).
C1578
lial 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.
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.
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 17, 2017
Thomas, E. J., S. E. Gabriel, M. Makhlina, S. P.
Hardy, and M. I. Lethem. Expression of nucleotide-regulated Cl⫺ currents in CF and normal mouse tracheal epithelial cell lines. Am J Physiol Cell Physiol 279: C1578–C1586,
2000.—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 cAMPactivating 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.
PURINERGIC ION TRANSPORT IN MOUSE TRACHEAL CELL LINES
MATERIALS AND METHODS
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 ␮g/ml), insulin (5 ␮g/ml), epidermal
growth factor (12.5 ng/ml), endothelial cell growth supplement (1.875 ␮g/ml), triiodothyronine (15 nM), hydrocortisone
(0.5 ␮M), 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 FITCconjugated 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, respec-
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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.
C1579
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PURINERGIC ION TRANSPORT IN MOUSE TRACHEAL CELL LINES
RESULTS
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. How-
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.
ever, 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.
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%
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tively. 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 ⍀ 䡠 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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PURINERGIC ION TRANSPORT IN MOUSE TRACHEAL CELL LINES
[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).
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 shortcircuit 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
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 ⱖ5 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].
small increases in the RT of 18.05 ⫾ 4.55 and 24.67 ⫾
8.67 ⍀ 䡠 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.
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 atten-
Table 1. Bioelectric properties of MTE18-(⫺/⫺) and MTE7b-(⫹/⫺) cell lines
⌬Isc, ␮A/cm2
Basal
MTE18-(⫺/⫺)
MTE7b-(⫹/⫺)
PD, mV
RT, ⍀° cm2
Isc, ␮A/cm2
Amiloride
cAMP
Ionomycin
0.56 ⫾ 0.04 (89)
0.86 ⫾ 0.08 (66)
165.0 ⫾ 9.0 (89)
323.0 ⫾ 20.0 (66)
3.24 ⫾ 0.18 (89)
2.04 ⫾ 0.14 (66)
⫺0.25 ⫾ 0.04 (72)
⫺0.16 ⫾ 0.04 (52)
0.0 ⫾ 0.0 (6)
0.0 ⫾ 0.0 (6)
8.50 ⫾ 1.51 (16)*
9.68 ⫾ 3.97 (12)*
Values are means ⫾ SE of number of cells in parentheses. Basal properties were obtained from cells mounted in Ussing chambers.
Responses to 10⫺4 M amiloride, cAMP (a cocktail of 10⫺5 M forskolin, 10⫺4 M IBMX, and 10⫺4 M dibutyryl cAMP), and 10⫺6 M ionomycin
are expressed as change in short-circuit current (⌬Isc). PD, potential difference; RT, transepithelial resistance. * P ⬍ 0.001, (Mann-Whitney
test).
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Fig. 2. Conditional immortalization of MTE cells shown as colonyforming 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 ⱖ10 experiments.
***P ⬍ 0.001 (Mann-Whitney test).
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PURINERGIC ION TRANSPORT IN MOUSE TRACHEAL CELL LINES
uated 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)
␮A/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).
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-re-
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.
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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 (䊐)
exposed to mucosal high-K⫹ –low-Cl⫺ Ringer solution. Values are
means ⫾ SE from ⱖ5 determinations.
sponse 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].
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 ␮M, with the
highest doses resulting in an increase in Isc of 28.2 ⫾
5.0 ␮A/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
PURINERGIC ION TRANSPORT IN MOUSE TRACHEAL CELL LINES
␮M thapsigargin causing a maximum inhibition of
96%.
DISCUSSION
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
⍀ 䡠 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.
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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 colonyforming 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
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PURINERGIC ION TRANSPORT IN MOUSE TRACHEAL CELL LINES
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
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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 DIDSinhibitable 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
PURINERGIC ION TRANSPORT IN MOUSE TRACHEAL CELL LINES
prove valuable in defining the potential role of CaCC in
the treatment of CF.
We are grateful to Dr. Elena Martsen for molecular biology expertise and guidance with RT-PCR.
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).
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