Calcium-stimulated Cl
secretion in Calu-3 human airway cells requires CFTR
Samina
Moon,
Meetpaul
Singh,
Mauri E.
Krouse, and
Jeffrey J.
Wine
Cystic Fibrosis Research Laboratory, Stanford University, Stanford,
California 94305-2130
 |
ABSTRACT |
Human airway
serous cells secrete antibiotic-rich fluid, but, in cystic fibrosis
(CF), Cl
-dependent fluid
secretion is impaired by defects in CF transmembrane conductance
regulator (CFTR) Cl
channels. Typically, CF disrupts adenosine 3',5'-cyclic
monophosphate (cAMP)-mediated
Cl
secretion but spares
Ca2+-mediated secretion. However,
in CF airway glands, Ca2+-mediated
secretion is also greatly reduced. To determine the basis of
Ca2+-mediated
Cl
secretion in serous
cells, we used thapsigargin to elevate intracellular Ca2+ concentration
([Ca2+]i)
in Calu-3 cells, an airway cell line bearing some similarities to
serous cells. Cells were cultured using conventional and air interface
methods. Short-circuit current
(Isc) and
transepithelial conductance
(Gte) were
measured in confluent cell layers. Thapsigargin stimulated large,
sustained changes (
) in
Isc and
Gte, whereas forskolin stimulated variable and smaller increases.
Isc was decreased by basolateral bumetanide, quinidine, barium, or
diphenylamine-2-carboxylate (DPAC) but was unaffected by high apical
concentrations of
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS),
4,4'-dinitrostilbene-2,2'-disulfonic acid, and calixarene. Isc was measured
after permeabilizing the basolateral membrane and establishing
transmembrane ion gradients. Unstimulated apical membranes displayed
high Cl
conductance
(GCl) that was
decreased by DPAC but not by DIDS. Apical
GCl could be
increased by elevating intracellular cAMP concentration but not
[Ca2+]i.
We conclude that CFTR channels are the exclusive
GCl pathway in
the apical membrane and display ~60% of maximum conductance at rest.
Thus elevated
[Ca2+]i
increases K+ conductance to force
Cl
through open CFTR
channels. We hypothesize that loss of CFTR channels causes diminution
of cholinergically mediated gland secretions in CF.
cystic fibrosis; Ussing chamber; epithelia; submucosal gland; cell
culture; amphotericin B; calcium ion; short-circuit current; chloride
ion; cystic fibrosis transmembrane conductance regulator
 |
INTRODUCTION |
SEROUS CELLS in human airways secrete antibiotic-rich
fluid (2). Serous cells in submucosal glands of the human lung (9) and
possibly in other regions of the distal airways (10) express high
levels of cystic fibrosis transmembrane conductance regulator (CFTR).
Primary cultures of submucosal gland serous cells also express CFTR and
secrete Cl
in response to
both cholinergic and adrenergic stimulation, with cholinergic
stimulation being more potent (36). In these cultures, agents stimulate
secretion to the extent that they elevate intracellular Ca2+ concentration
([Ca2+]i;
see Ref. 37). For example, isoproterenol but not forskolin elevated
[Ca2+]i,
and isoproterenol but not forskolin was effective in stimulating short-circuit current
(Isc; see Ref.
37). In cultured gland cells from cystic fibrosis (CF) subjects,
responses to all mediators, including those that elevate
[Ca2+]i,
are greatly reduced (16, 34, 35). The diminution of Ca2+-mediated secretion in CF
distinguishes submucosal gland cells from other organs such as sweat
glands (22), and many organs in the mouse (8) in which
Ca2+-mediated responses are
altered little in the CF phenotype. However, it is consistent with the
loss of Ca2+-mediated secretion in
intestinal crypt cells of CF subjects (5).
Three general mechanisms might explain the reduction of
Ca2+-mediated secretion in CF
tissues (Fig. 1). 1)
CFTR might be activated by a
Ca2+-dependent mechanism (4, 29).
2) CFTR might be required to activate a different,
Ca2+-dependent
Cl
channel.
3) CFTR might be the predominant
apical Cl
conductance
(GCl)
pathway and might also be substantially activated in unstimulated
cells. If both conditions were met in the third mechanism, elevated
[Ca2+]i
could stimulate secretion by activating basolateral
K+ channels and the
bumetanide-sensitive
Na+-K+-2Cl
cotransporter (14). Both of these effects would be eliminated if
CFTR-mediated apical
GCl were absent
(23, 24).
The hypothesis to be tested in this paper is whether the bulk of
cholinergically stimulated,
Cl
-mediated fluid secretion
by human lung serous cells uses mechanism 3 in which CFTR channels, open at rest, serve as the
exclusive conductance pathway for
Cl
exit across the apical
membrane and increased
[Ca2+]i
opens basolateral K+ channels to
drive Cl
through the open
CFTR channels.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
Three hypothetical mechanisms for cystic fibrosis transmembrane
conductance regulator (CFTR)-dependent,
Ca2+-stimulated
Cl secretion.
A: CFTR is known to be influenced by
protein kinase C (PKC; see Ref. 29). This effect could be a major
activating pathway in some cell types.
B: CFTR is known to influence other
ion channels (27). An alternate
Cl channel in Calu-3 cells
could require both Ca2+ and CFTR
to function. C: If CFTR were normally
open at rest, elevation of Ca2+
could produce secretion by opening basolateral
K+ channels to provide a driving
force for apical Cl exit.
[Ca2+]i,
intracellular Ca2+
concentration.
|
|
To test this hypothesis, we used Calu-3 human airway cells, which have
many similarities to submucosal gland serous cells (11, 15, 23). Calu-3
cells express a high level of CFTR, develop tight junctions, and
secrete Cl
via apical CFTR
channels. An analysis of their behavior using various pharmacological
agents and nystatin-permeabilized cell sheets indicates that ~75% of
the maximal apical
GCl is already present in unstimulated cells and that the control of secretion is then
driven by increases in cytosolic free
Ca2+, which hyperpolarizes the
basolateral membrane (23). However, because the agents used previously
caused only transient increases in
Isc (23), it
remains possible that more sustained elevations in
[Ca2+]i
could activate CFTR (mechanism 1) or
a different apical Cl
channel that is CFTR dependent (mechanism
2). Therefore, we have investigated the mechanism of
Ca2+-mediated
Cl
secretion in Calu-3
cells using thapsigargin to achieve sustained increases in
[Ca2+]i
without activation of other messengers that might actually truncate
responses (17).
 |
METHODS |
The Calu-3 cell line was obtained frozen from the American Type Culture
Collection (Rockville, MD). After being thawed, the cells were placed
in tissue culture flasks (Costar, Pleasanton, CA) containing
Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and
penicillin-streptomycin and were grown at 37°C in an atmosphere of
5% CO2-95% air. Confluent
monolayers were subcultured by trypsinization with a solution of
phosphate-buffered saline (PBS), 0.04% EDTA wash, and 0.25% trypsin.
Cells were passaged in T75 flasks at a 1:4 dilution or
plated at 106
cells/cm2 onto Costar Transwell
inserts (0.45-µm pore size,
0.5-cm2 surface area; Costar,
Cambridge, MA) coated with human placental collagen. After being
plated, cells were maintained in culture at least 6 days before use,
and medium was changed every 2-3 days.
We compared two kinds of culture conditions. In conventional
("submersed") culturing, medium was added to both sides of the filter. In air interface culturing, medium was added only to the basolateral side of the inserts. Air interface culturing markedly improves the differentiation of primary cultures of many kinds of
epithelia (see Ref. 25 for further discussion).
Standard techniques were used in Ussing chamber studies. Filters on
which cells had grown to confluency were cut from the plastic inserts
and mounted between half-chambers so that they separated mucosal and
serosal bathing solutions of identical ionic composition. Mean values
for transepithelial conductance
(Gte) in our
experiments were 15 ± 4 mS/cm2
for air interface cultures (n = 11)
and 7 ± 1 mS/cm2
(n = 25) for submersed cultures. In
recent pilot experiments using confluent cells on intact
human placental collagen-coated Snapwell filters,
Gte was decreased to 6 mS/cm2 for air interface cultures
(n = 11) and 2 ± 1 mS/cm2 for submersed cultures
(n = 7). However, these
monolayers also show reduced responses to mediators compared with what
was observed in this series of experiments (C. Penland and M. Lee,
unpublished observation). Both sides of the monolayers were bathed with
12 ml of Krebs-Henseleit solution, which contained (in mM) 128 NaCl, 4.6 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4,
25 NaHCO3, and 11.2 glucose;
osmolarity was adjusted to 320 mmol/l, and pH was 7.4 when
gassed with 95% O2-5%
CO2 at 37°C. Drugs were
added in small volumes from concentrated stock solutions. Gas-lift
oxygenators ensured proper oxygenation and stirring of the solutions.
The transepithelial potential difference
(Vte) was
measured with agar bridges connected through calomel half-cells to a
high-impedance electrometer. An external circuit used to bring the
Vte to zero was
connected to the backs of the half-chambers via agar bridges. The
amount of electric current needed to maintain this voltage clamp was
measured continuously on a chart recorder and on an LCIII computer
using MacLab interface and software. Because the membrane was clamped
to zero potential, it was regarded as short circuited, and the current
that flowed under these conditions was called
Isc. This
Isc was
considered to be representative of all the transport processes actively
occurring across the tissue. Gte was estimated
at 2-s intervals by measuring current changes in response to 1-mV
pulses. Data are presented as means ± SE.
More direct measurements of apical membrane
GCl were made by
permeabilizing the basolateral membrane with amphotericin B (100 µM).
This level of amphotericin B was determined as the concentration at
which no response to bumetanide was seen (12). We then established an
11:1 serosal-to-mucosal ionic gradient for
Cl
by bathing the
basolateral surface in a
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-based solution containing 140 mM
Cl
and the apical surface
in an identical solution except for the replacement of 91% of the
Cl
with gluconate or
aspartate.
All chemicals were reagent grade and, unless otherwise specified, were
obtained from Sigma Chemical (St. Louis, MO). Stock solutions of
forskolin (Calbiochem, La Jolla, CA) in ethanol, calixarene (a gift
from R. Bridges and A. K. Singh) in water, and thapsigargin in dimethyl
sulfoxide (DMSO) were stored at
20°C. Stock solutions of
bumetanide in DMSO and ouabain in water were stored at 4°C.
4,4'-Dinitrostilbene-2,2'-disulfonic acid (DNDS; obtained
from Pfaltz & Bauer) and
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)
were dissolved in water. Fresh stocks were prepared daily for
diphenylamine-2-carboxylate (DPAC) in DMSO. Fetal bovine serum was
obtained from Hyclone.
 |
RESULTS |
Growth conditions had a marked effect on
responses. Calu-3 cells grown at the air interface
differ from submersed cultures in many ways. For example, air interface
cultures have much larger basal
Isc and lack the
large, spontaneous drifts in
Isc that are observed in many submersed cultures (25). The present experiments revealed the following general differences resulting from the two
culture conditions.
Thapsigargin, which is membrane permeant, produced short-latency,
fast-rising, multicomponent responses when applied to the apical side
of monolayers grown as submersed cultures, but, to our surprise, it was
ineffective on the basolateral side of submersed cultures. Thapsigargin
was effective only when applied to the basolateral surfaces of air
interface cultures, but the responses were long latency, slowly rising,
and single component. The sidedness of thapsigargin stimulation was
noted in the first study in which it was applied to native epithelia
(6) but remains unexplained. Responses to forskolin also differed as a
result of culture conditions. In submersed cultures, responses to
forskolin were absent or small. For monolayers grown at the air
interface, forskolin caused an average increase in
Isc of ~40
µA/cm2, but this was still much
smaller than responses to thapsigargin.
Despite these differences, we will propose a common model for
Ca2+-stimulated
Cl
secretion in cells grown
in both kinds of conditions.
Agents that elevate
[Ca2+]i
produced large increases in
Isc.
Carbachol and histamine caused transient increases in
Isc (Fig.
2 and Ref. 23). Basolateral application of
carbachol caused responses that peaked within 5 s and fell to
half-maximum within 16 s. The average peak responses to 10 or 100 µM
carbachol were 17 and 77 µA/cm2,
respectively, with a large amount of variability (Fig.
2C). For 100 µM carbachol, the
average conductance increase at the peak of the response was 87 ± 38% (n = 9). Subsequent application of carbachol produced a response that was <10% of the initial response (n = 4). Basolateral
application of 10 µM histamine caused an average peak increase of 7 µA/cm2
(n = 5), with a time course and
variability similar to those observed after carbachol. Responses to
carbachol and histamine were abolished by pretreatment with bumetanide
(n = 3) or
BaCl2 (n = 2), indicating that they are
Cl
secretory responses that
depend on both the bumetanide-sensitive Na+-K+-2Cl
cotransporter and Ba2+-sensitive
basolateral K+ channels. Carbachol
and histamine are known to elevate
[Ca2+]i,
but the intracellular messengers that they liberate can have additional
effects (17). Therefore, we used thapsigargin to obtain elevated
[Ca2+]i
without additional effects of other intracellular messengers.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Transient short-circuit current
(Isc) responses
to carbachol. Trace shows the largest
Isc increase
obtained with basolateral application of 100 µM carbachol. Vertical
deflections on trace (see insets A and
B) are responses to 1-mV pulses used
to measure transepithelial conductance
(Gte).
Stimulation with 10 µM forskolin did not increase either
Isc or
Gte in this
monolayer. Inset C: range of responses
to carbachol observed in 9 monolayers. Filled circles are submersed
cultures prestimulated with forskolin, filled squares are submersed
cultures not prestimulated with forskolin, and open squares are air
interface cultures not prestimulated with forskolin. In this and all
following Isc
traces, difference between baseline current and zero represents basal
Isc. Properties
of basal Isc were
described in Ref. 25.
|
|
Thapsigargin selectively inhibits the
Ca2+ pump of the endoplasmic
reticulum to produce modest but sustained elevations in
[Ca2+]i
(31) and hence is useful for demonstrating a pure effect of
[Ca2+]i
on ion transport (7, 17). Thapsigargin caused large increases in
Isc and
Gte in almost all
Calu-3 monolayers (Figs. 3 and
4). Figure 3 is an example of the
short-latency (<10 s), biphasic responses that were typical for
monolayers grown as submersed cultures. The early portion of the
initial transient fell rapidly (time to half-amplitude ~3 min,
n = 4). The second component was much
more sustained, with a mean time to half-maximum of 37 ± 7 min
(n = 13). The average peak changes in
conductance after thapsigargin ranged from 52 to 117% in different
conditions (Table 1).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Isc responses to
apical thapsigargin in submersed cultures. Each trace shows
Isc across a
monolayer of Calu-3 cells. Line thickness is caused by the voltage
pulses used to measure conductance (see Fig. 2), which can not be
resolved when prolonged recordings are compressed.
A: stimulation with thapsigargin (300 nM, apical) caused a large increase in
Isc and
conductance and rendered the monolayer refractory to further
stimulation with Ca2+-elevating
agents. Stimulated
Isc was only
transiently affected by dimethyl sulfoxide (DMSO) vehicle (V) but was
abolished by 200 µM bumetanide (Bm). Ba, barium. Marks near end of
trace mark additions of compounds of no relevance.
B: preaddition with bumetanide (10 and
100 mM) caused marked inhibition of response to thapsigargin.
C: lack of response to forskolin (10 µM) and failure of
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 200 mM) and calixarene (30 nM), both potent blockers of outwardly
rectifying Cl channels, to
inhibit thapsigargin-stimulated
Isc.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
Isc responses to
basolateral thapsigargin and various inhibitors in monolayers grown at
air interface. A: response to 300 nM
thapsigargin applied basolaterally, followed by apical calixarene (30 nM), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS; 3 mM), DIDS (400 µM; vertical lines), which had no effect, and
diphenylamine-2-carboxylate (DPAC; 3.2 mM) added to both chambers,
which abolished the response. B:
response to 300 nM thapsigargin applied basolaterally, followed by 10 µM basolateral bumetanide. C:
forskolin (10 µM, added at origin) produced a small increase in
Isc that is not
further increased by apical thapsigargin (300 nM, first vertical line),
but basolateral thapsigargin caused a large response that was
unaffected by apical application of calixarene, DNDS, and DIDS
(vertical lines, same concentration as above).
Isc was abolished
by 3.2 mM DPAC added to both chambers.
|
|
After thapsigargin, all other
Ca2+-elevating agonists were
ineffective. For example, Fig. 3A
shows that a second addition of thapsigargin or two applications of
carbachol evoked no further responses. Stimulated
Isc was
only transiently affected by DMSO vehicle (Fig.
3A).
The responses to thapsigargin were primarily mediated by basolateral
Na+-K+-2Cl
cotransport because they were inhibited by bumetanide. When 10 µM
bumetanide was added after thapsigargin, the change (
) in Isc to
thapsigargin was reduced by 69 ± 5% for submersed cultures (n = 10) and by 82 ± 5% for air
interface cultures (n = 15; Fig. 3A). When bumetanide was added
before thapsigargin, the response was reduced by 88 ± 8% for both
types of cultures (n = 12). Bumetanide did not reduce
Gte. These
results differ markedly from the small effect of bumetanide on basal
Isc (25).
Response latencies (time to first rise) to basolateral thapsigargin in
air interface cultures were ~30 times longer (~5 min) than to
apical addition of thapsigargin in submersed cultures and lacked the
initial peak seen in submersed cultures (compare Figs. 3 and 4). At
least part of the markedly slower latency seen in air interface
responses can be attributed to the lack of the early fast component of
the response.
Oscillating responses to thapsigargin sometimes occurred in monolayers
grown in either condition (Fig. 3). Such oscillations suggest that
[Ca2+]i
is oscillating with a similar rhythm in the majority of cells (13), but
the mechanism whereby changes in
[Ca2+]i
are coordinated among the multiple cells
(~106 cells) in the monolayer is
unknown.
DIDS, usually considered a blocker of
Cl
channels and exchangers
(see below), in fact produced large, sustained increases in
Isc when applied
basolaterally to submersed cultures of Calu-3 cells, with a
half-maximal effective concentration of ~120 µM (Fig. 5). (DIDS was not tested on the
basolateral surface of air interface cultures.) Similar anomalous
increases in Isc
to basolateral DIDS were reported previously for
T84 colonic tumor cells (7). In
T84 cells, the responses were shown to result
from increases in
[Ca2+]i.
The same mechanism is likely in Calu-3 cells because bumetanide inhibition of responses to DIDS was equivalent to its inhibition of
thapsigargin responses (68%, n = 4) and because other
Ca2+-elevating agents were
relatively ineffective after basolateral DIDS. How DIDS elevates
[Ca2+]i
is not known (see Ref. 7 for discussion), but, like thapsigargin and
unlike most agonists, DIDS causes sustained increases in
Isc.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Basolateral DIDS produces large increase in
Isc. Apical
application of carbachol (100 µM) caused a short-latency transient
response, but little or no responses were produced by apical
isoproterenol (Iso, 100 µM) or 10 and 100 µM DIDS. However, 200 µM DIDS applied basolaterally produced a large, short-latency
response. Monolayer had been grown in submersed culture for 13 days
before testing. Inset shows
dose-response relation for basolateral DIDS.
EC50, half-maximal effective
concentration; Conc, concentration; Max, maximal.
|
|
We also attempted to elevate Ca2+
with ionomycin, but with escalating concentrations, we found that
responses at threshold (0.2 µM) led to large, biphasic swings of
Isc and
irreversible, continuous increases in
Gte within 5 min.
Elevation of intracellular adenosine 3',5'-cyclic
monophosphate concentration usually produced small increases in
Isc.
Forskolin produced much smaller responses than did thapsigargin. For
submersed cultures, forskolin failed to stimulate
Isc in 9 of 14 preparations (e.g., Fig. 3C), and
the mean
Isc
for the 4 responding preparations was only 16 ± 6 µA/cm2. For air interface
cultures, the mean
Isc to
forskolin was 39 ± 12 µA/cm2
(n = 7), and there was a tendency for
older cultures to develop larger responses to forskolin. Although this
level of response would be considered robust in many preparations, it
is only 23% of the
Isc to
thapsigargin in these same preparations.
In 9 of 13 submersed cultures, forskolin also produced only small
responses when applied after thapsigargin; the mean response to
forskolin after thapsigargin in these nine monolayers was only 8 ± 3 µA/cm2. These small responses
to forskolin are consistent with the hypothesis that basolateral
K+ conductance
(GK) is usually
limiting for Isc
in Calu-3 cells and that apical CFTR channels are normally open
(hypothesis 3; see Ref. 23).
Exceptions to general results with thapsigargin and
forskolin. We encountered several marked exceptions to
the general findings that thapsigargin produces large increases in
Isc and that
forskolin produces small increases. In air interface cultures, the
largest responses to forskolin approached the amplitude of responses to thapsigargin and oscillated with the same period as oscillations produced by thapsigargin (Fig. 6). In
submersed cultures, exceptionally large responses to forskolin (Fig.
7) were observed after unusually small
responses to thapsigargin in three experiments. The mean response to
forskolin in these experiments was 147 ± 26 µA/cm2, a value ~20 times the
mean for all forskolin responses and equivalent to the average response
to thapsigargin. A model to explain both typical and exceptional cases
is presented in the DISCUSSION.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Atypical large responses to forskolin. Two of the largest responses to
forskolin observed in air interface cultures are shown.
A: change ( ) in
Isc was abolished
by 3.2 mM DPAC added to both sides. Compare these responses with the
much smaller responses to forskolin or isoproterenol shown in Figs.
2-5. B: corresponding responses
to thapsigargin from a paired filter. Time 0 was
set at point of agonist addition; prior baseline was stable at the
levels shown. Voltage deflections used to measure conductance were
removed from these traces. Top trace in
B is same data as in Fig.
4A but was rescaled for comparison
with forskolin responses. ms572R, ms572L, ms573R, and ms573L indicate
experiment numbers.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 7.
Atypical small responses to thapsigargin and subsequent large responses
to forskolin. In each of these submersed cultures, thapsigargin
produced atypical small responses without a fast component. In
A the response to thapsigargin was
20% of average; in B, the response
was negligible. Subsequent application of forskolin produced
exceptionally large, fast responses. This response pattern was observed
in 3 of 13 experiments with submersed cultures and was never seen with
air interface cultures.
|
|
Effects of inhibitors are consistent with CFTR-mediated,
K+-limited
Cl
secretion.
CFTR is unusual among known epithelial
Cl
channels with regard to
its insensitivity to Cl
channel blockers. Swelling-activated (26) and some
Ca2+-activated
Cl
channels are blocked by
stilbenes, but CFTR is not. Outwardly rectifying,
depolarization-activated Cl
channels (ORDIC channels) that are often seen in excised
patches are also blocked by DNDS (12, 26), DIDS (26), and calixarene. Thus these compounds can be used to determine if any of these channels
contribute to Isc
in Calu-3 cells. We applied DIDS (up to 600 µM), DNDS (up to 2 mM),
and calixarene (30 nM) in various combinations to the apical surface of
cells with no significant effect on basal
Isc or
Isc stimulated
by thapsigargin, forskolin, or their combination (Figs. 3-5 and
Table 2).
In contrast to these negative results, responses to thapsigargin in
both submersed and air interface cultures were reduced by basolateral
applications of bumetanide, an inhibitor of basolateral Na+-K+-2Cl
cotransport (Figs. 3 and 4), by DPAC added to either side
(Figs. 4 and 6), and by basolateral application of the
K+ channel blockers
BaCl2 or quinidine. Results with
various inhibitors are quantified in Table 2 and Fig.
8.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 8.
Inhibition of
Isc by channel
blockers and transport inhibitors. Each bar shows mean %inhibition ± SE. Concentrations, no. of cultures, and exact numerical values
for Isc and
Gte are given in
Table 2. All effects were stable except for
BaCl2, which precipitated from the
solution. For BaCl2, the
%inhibition is the peak of the transient inhibition observed.
|
|
Spontaneous increases in
Isc.
All Calu-3 cultures, no matter how grown, had a significant basal
Isc mediated
mainly by bumetanide-insensitive,
-dependent Cl
secretion, with a
smaller component of electrogenic sodium-glucose transport (25).
However, in addition to the basal
Isc, ~40% of
submersed cultures (vs. 0% of air interface cultures) spontaneously developed large increases in
Isc within 30 min
after being placed in the Ussing chamber. The properties of the
spontaneous increases in
Isc suggest that
they represent Cl
secretion
caused by increases in
[Ca2+]i
because they are inhibited by bumetanide (25) and because subsequent
responses to thapsigargin are reduced by 88 ± 9%
(n = 9).
Experiments on cell sheets with permeabilized basolateral membranes
and transepithelial Cl
gradients.
Results to this point show that agents that increase
[Ca2+]i
produce large increases in
Isc and that
forskolin is usually much less effective. Possible explanations for the
efficacy of thapsigargin were outlined in Fig. 1. The small or absent
Isc in
response to forskolin could arise if apical CFTR
Cl
channels are
constitutively active and if electrochemical equilibrium for
Cl
across the apical
membrane is not altered. In these cases, vectorial Cl
movement would be
controlled indirectly by the basolateral exit pathway for
K+.
To help decide among these possibilities, we determined the effect of
different agonists on apical
GCl by
establishing Cl
or NaCl
gradients across the monolayers and then permeabilizing the basolateral
membrane with amphotericin B. Because of the frequent, spontaneous
increases in Isc
observed in submersed cultures (25), we only used the more stable air
interface cultures for these experiments.
For basolaterally permeabilized monolayers ("apical membrane"
preparations), with a 11:1 gradient of
Cl
, addition of
thapsigargin caused no change in
Isc
(
Isc = 0.8 ± 0.4 µA/cm2,
n = 13) or
Gte
(
Gte = 0.2 ± 0.1 mS/cm2), whereas
forskolin stimulated a significant
Isc of 33 ± 7 µA/cm2 and
Gte = 1.8 ± 0.6 mS/cm2
(n = 9; Fig. 9). These
results extend previous results using different methods of
permeabilization and transiently acting
Ca2+ agonists (23) and reinforce
the conclusion that the apical membrane of Calu-3 cells lacks
Ca2+-activated
Cl
channels.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 9.
Apical membrane of Calu-3 cells lacks
Ca2+-activated and
stilbene-sensitive Cl
channels. Inset shows experimental
arrangement: the monolayer of Calu-3 cells was placed in an 11:1
serosal-to-mucosal Cl
gradient, and ouabain and amphotericin B were applied to the
basolateral membrane. Electrical recording of
Isc was mainly
generated by Cl diffusion
potential across apical membrane and paracellular pathway and
Gte (trace
thickness, see Fig. 2). Trace starts immediately after application of
amphotericin B. Of agents applied, only forskolin and DPAC affected
transapical Isc
and Gte. Four
vertical lines before DPAC mark additions of 30 nM calixarene, 400 µM
DIDS, and 0.3 and 3 mM DNDS, respectively.
|
|
To assess the proportion of apical
GCl that is
active before stimulation, we established 11:1 gradients of
Cl
, permeabilized the
basolateral membrane, applied forskolin to maximize the apical
GCl, and finally
applied DPAC to eliminate apical
GCl. The maximum
Isc and
Gte observed
after forskolin was set to 100%, and the value after DPAC was set to
0%. The average proportion of
Isc before
application of forskolin was then measured and was determined to be 57 ± 5% (range 30-76%; n = 10;
Fig. 10A). The
average proportion of
Gte before
application of forskolin was 50 ± 10%
(n = 6; Fig.
10B; see also Ref. 23).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 10.
CFTR channels in apical membrane are active before stimulation. Using
the same experimental arrangement, we determined the proportion of CFTR
channels active before stimulation by setting the peak
Isc and
Gte after maximal
forskolin to 100% and the minimum after maximal DPAC to 0%.
A: example of a preparation with a low
level of active CFTR channels at rest, measured via
Isc.
B: example of a preparation with a
high level of CFTR activity at rest measured via
Gte.
|
|
To determine the actual apical membrane
GCl and the tight
junction plus leak permeability, we assumed that DPAC eliminates apical
GCl without
affecting tight junction conductance and that the apical membrane has
no appreciable conductance for cations. Given these assumptions, the
difference in conductance between permeabilized monolayers before and
after treatment with DPAC is a measure of apical membrane
GCl, and the
residual conductance is attributable to paracellular pathways,
nonspecific leak pathways caused by edge damage, and incomplete
inactivation of CFTR by DPAC. We obtained an average value of 8 mS/cm2 for apical
GCl and 13 mS/cm2 for paracellular plus leak
conductance (n = 24; Fig.
11).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 11.
Dose-response curves for inhibition of
Isc and
Gte by DPAC. Curves have identical shapes with
half-maximal effective concentration (500 µM) and a Hill slope of 1. This implies that the apical conductance is through a single type of
Cl channel.
|
|
 |
DISCUSSION |
A model of
Ca2+-stimulated
Cl
secretion by Calu-3 cells.
In the simple model outlined in Fig. 12, we hypothesize
that Cl
secretion is
controlled by just two switches, the apical
Cl
(CFTR) and the
basolateral K+ channel
populations. The activity of these two channel populations vary
independently and are controlled by intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration and
[Ca2+]i,
respectively. In Calu-3 cells grown at air interface, the apical CFTR
conductance is ~60% of maximum at rest. The key features of
the model are that 1) CFTR is the
only apical Cl
channel and
2) a
Ca2+-activated
GK in the
basolateral membrane is the key determinant of secretion. Basal
secretion does not require
Na+-K+-2Cl
cotransport, but stimulation recruits this transporter by an unknown
mechanism (25). The major features of this model are consistent with
previous models of Calu-3 cells (15, 23), with human submucosal gland
cells (35, 37, 38), and with human tracheal epithelium
(24).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 12.
Model of Ca2+-stimulated
Cl secretion by Calu-3
cells. Basolateral membrane is at
left. Key features of the model are
that 1) CFTR is the only apical
Cl channel;
2) basolateral membrane contains a
Ca2+-activated
K+ channel but no cAMP-activated
K+ conductance;
3) a substantial proportion of CFTR
channels are active at rest; and 4)
stimulation recruits
Na+-K+-2Cl
cotransport by an unknown mechanism. Our experiments do not specify the
mechanism(s) of increased apical Cl conductance or
basolateral K+ conductance, which could result from
increased no. of channels (via vesicle insertion), increased open
probability, or both.
|
|
Three findings support the hypothesis that CFTR is the only apical
Cl
channel operating in
Calu-3 cells. First, thapsigargin produced no change in apical
GCl. Second,
DIDS, DNDS, and calixarene caused no inhibition of apical
GCl. Third,
patch-clamp experiments from apical membranes of confluent Calu-3 cells
show that CFTR channels accounted for virtually all
Cl
channels observed (15).
It remains possible that the apical membrane contains a non-CFTR
Cl
channel, but, if so, the
properties of that channel are considerably constrained by our results.
Such a channel must be normally open, not sensitive to
Ca2+, not inhibited by DIDS, DNDS,
or calixarene, and not detectable with patch-clamp methods. It is also
possible that a Ca2+-dependent
channel exists but is either fully activated or fully inhibited by
treatment of the basolateral membrane with amphotericin B.
We used DPAC to inhibit apical
GCl. DPAC is
often used to reduce or eliminate conductances mediated by CFTR, but
DPAC is not a specific blocker of CFTR. Although it does cause an open
channel block (25), it also severely reduces intracellular cAMP
concentration levels by an unknown mechanism (18, 33). The inhibition
of Isc and
GCl that we
observed probably represents a combination of channel block and
reduction of intracellular cAMP concentration. This would not
necessarily alter our conclusion, but the model is weakened by the
possibility that DPAC is having still other unknown effects. However,
sodium-glucose transport is not inhibited by DPAC, and
Gte is decreased
rather than increased; hence, monolayer integrity, the sodium-glucose
transporter, and
Na+-K+-ATPase
activity would seem to be unaffected by high levels of DPAC.
If it is provisionally accepted that apical
GCl is determined
by CFTR, then evidence that CFTR channels are active in unstimulated preparations is based on the high levels of basal
Isc seen here and
in other studies (23, 25), the high apical
GCl observed in
unstimulated, basolaterally permeabilized preparations (Fig. 10, Table
1, and also Ref. 23), and the activity of single CFTR channels in
unstimulated Calu-3 cells (15). We attempted to quantify the proportion
of maximal (forskolin-stimulated) apical GCl that was open
at rest by the methods outlined in Fig. 10. This method is accurate
only to the extent that DPAC eliminates
GCl. Because it
is not certain that DPAC eliminates 100% of apical GCl, our estimate
that 60% of maximal apical
GCl is active at rest is a minimum estimate. Because DPAC eliminates all
Cl
secretion in intact
preparations (Table 2), we hypothesize that the remaining
GCl arises from
the paracellular and leak pathways. Our estimate that 60% of maximal
GCl is active in
basal conditions will be inaccurate to the extent that the basolateral
membrane is not fully permeabilized by amphotericin B, the apical
membrane is partially permeabilized by amphotericin B, or the
paracellular pathway is affected by stimulation or by
blockers.
Four observations support the hypothesis that basolateral
GK is
Ca2+ activated.
1) Thapsigargin, which elevates
[Ca2+]i
without elevating other intracellular messengers, is the most potent
stimulus we have found for stimulating
Isc.
2) Thapsigargin had no effect on
apical GCl but
did markedly increase
Gte.
3) Basolateral
Ba2+ inhibited the thapsigargin
stimulation of
Isc.
4) Forskolin often had little or no
effect on secretion, suggesting that a cAMP-activated GK is either
absent or is a minor, fully active component of
GK in Calu-3
cells.
Although simple, the model can account for both the typical and
atypical responses that we observed. The modal response for Calu-3
cells was a small response to forskolin and a large response to
thapsigargin, with an average response to the combined agents that was
only slightly larger than to thapsigargin alone. These results indicate
that cAMP doesn't increase basolateral
GK in Calu-3
cells because if it did forskolin should always produce large
responses. More than 90% of our preparations fit the above description, but, as shown in Table 3, we
also encountered monolayers that behaved as if both channels were
closed (initially refractory to either agent), as if both channels were
open (spontaneous increases in
Isc seen only in
submersed preparations), and as if
K+ channels were active but CFTR
was relatively inactive (small response to thapsigargin but large
response to forskolin).
View this table:
[in this window]
[in a new window]
|
Table 3.
Four possible configurations of apical and basolateral channels and
predicted consequences or responses to elevations of
[Ca2+]i and [cAMP]i
|
|
Comparison of models of Cl
secretion by Calu-3 cells and T84 cells.
The proposed mechanism for
Cl
secretion in Calu-3
cells shares features with T84 cells, which
are a model for colonic crypt cells. In T84
cells, most evidence is consistent with the hypothesis that confluent
sheets of polarized cells have CFTR as the primary or exclusive apical
GCl (30), and
many Isc
experiments were interpreted to mean that
Ca2+-mediated secretion resulted
from activation of Ca2+-dependent,
basolateral K+ channels (7, 17),
although in at least one experiment an additional DIDS-sensitive
conductance was observed (20). A major difference between
T84 and Calu-3 cells is the extent to which CFTR channels are open in Calu-3 cells. A possible difference is that
T84 cells may have a significant
cAMP-activated basolateral GK (28), whereas
Calu-3 cells do not. Finally, unlike Calu-3 cells,
T84 cell
Isc is entirely
inhibited by bumetanide.
How well do Calu-3 cells model human submucosal gland serous cells?
Human gland serous cells are relatively inaccessible, and the study of
Calu-3 cells is just beginning, so comparisons are necessarily limited.
In the only single-channel patch-clamp study of human submucosal gland
serous cells, channels having the properties of CFTR channels were
observed, but Ca2+-dependent
Cl
channels were not (3). In Ussing chamber comparisons
of gland cells from normal and CF subjects, it was found that
Cl
secretion in CF cells
was greatly reduced, not only to agents that elevated intracellular
cAMP concentration but also to agents that elevated
[Ca2+]i,
consistent with the proposed model (35).
Several recent reports might be taken as evidence against the model and
in favor of Ca2+-dependent
Cl
channels in human
submucosal gland serous cells. Yamaya et al. (38) found that
Cl
secretion by human gland
cells could be stimulated by purinergic agents and the responses
inhibited by DIDS. Although DIDS inhibition might be considered
suggestive of a non-CFTR Cl
channel, it seems more likely that DIDS was acting as an antagonist of
the P2Y receptor (19). A series of
papers using feline submucosal gland serous cells has presented
evidence for Ca2+-dependent
Cl
channels in that species
(e.g., Ref. 21). Although human preparations were also studied
sometimes, all records presented are from cats. In summary, we know of
no compelling evidence for
Ca2+-dependent
Cl
channels in human
submucosal gland cells. Thus Calu-3 cells and human submucosal gland
serous cells may be similar both biochemically (11) and
electrophysiologically, but much additional work on both types of cells
is necessary.
Implications for CF. We hypothesize
that Calu-3 cells are a model for submucosal gland serous cells (11,
15, 23). If true, it follows that CF serous cells will fail to secrete
to cholinergic agents (35) because such secretion depends on apical CFTR channels that are open at rest (15, 23, 25). Our present understanding of submucosal gland function is that CFTR expression is
high in serous cells and low or absent in mucous cells (9). Hence, in
CF, the hypothesis is that submucosal glands will secrete mucus that is
not hydrated by antimicrobial-rich fluid from serous cells. A similar
imbalance between fluid and mucus secretion may play out in the small
airways where serous and mucous cells are located at the surface. The
evaluation of this hypothesis, and other hypotheses about salt
composition of airway fluid, will require new methods and perhaps new
model systems.
It is often stated that cAMP-mediated secretion is defective in CF but
that Ca2+-mediated secretion is
intact. This claim is the basis for therapies designed to circumvent CF
symptoms by activating latent or underutilized Ca2+-activated
Cl
channels (alternate
Cl
channels) in the lungs
and other organs affected in CF. The generalization is reinforced by
evidence that CFTR Cl
channel activity requires phosphorylation by cAMP-dependent protein kinase, with less important roles being played by
Ca2+-activated kinases. However,
in human colonic epithelium, CF causes a loss of
Cl
secretion that is
stimulated by both Ca2+ and
cAMP-mediated pathways (5, 32). Human lung submucosal gland cells also
show a severe reduction in
Ca2+-mediated secretion in CF (16,
35).
Calu-3 cells are not a good model for most airway surface epithelial
cells, which, at least in the upper airways, contain low levels of CFTR
but higher levels of epithelial
Na+ channel. Surface cells of the
upper airways are primarily involved in absorption of
Na+,
Cl
, and fluid (34), whereas
submucosal gland serous cells are primarily involved in fluid
secretion. Airway surface cells can be stimulated to secrete, and,
unlike Calu-3 cells, surface cells contain
Ca2+-activated
Cl
channels (1, 8).
An emerging theme in CF research is that, in both mice and humans,
organs that contain alternative
GCl do not
develop CF disease (8), suggesting either that
GCl is the
critical function that CFTR performs or that alternative
Cl
channels can also mimic
other functions of the CFTR. But this creates an enigma: if human
airway surface epithelia contain an abundant, alternate
GCl, why are the
lungs subject to CF disease? Our hypothesis avoids that enigma: the
loss of apical
GCl in submucosal gland serous cells is critical to human CF lung disease because these
cells lack alternate Cl
channels. The predicted consequence is a reduction in secretions of
antibiotic-rich fluids that are thought to be a crucial component of
airway mucosal defenses (2, 11, 15, 23, 25, 35).
The implication of this hypothesis is discouraging because it suggests
that the strategy of activating "bypass"
Cl
channels may not work
for submucosal gland serous cells. However, it remains possible that
activation of alternate Cl
channels in surface epithelia might prevent or at least slow the course
of CF lung disease.
 |
ACKNOWLEDGEMENTS |
We thank Tina Law, Ilynn Nepomuceno, and Clare Robinson for cell
culture, R. Bridges and A. K. Singh for a gift of calixarene, and Chris
Penland for suggestions on the manuscript.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
HL-42368 and DK-51817, Cystic Fibrosis Research, Inc., Cystic Fibrosis
Foundation, Ron and Kay Presnell, and Patricia Bresee. S. Moon and M. Singh were recipients of a Cystic Fibrosis Foundation Student
Traineeship and a Howard Hughes Summer Research Fellowship.
Address for reprint requests: J. J. Wine, Cystic Fibrosis Research
Laboratory, Bldg. 420 (Jordan Hall), Stanford University, Stanford, CA
94305-2130.
Received 11 February 1997; accepted in final form 21 August 1997.
 |
REFERENCES |
1.
Anderson, M. P.,
D. N. Sheppard,
H. A. Berger,
and
M. J. Welsh.
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L1-L14,
1992[Abstract/Free Full Text].
2.
Basbaum, C. B.,
B. Jany,
and
W. E. Finkbeiner.
The serous cell.
Annu. Rev. Physiol.
52:
97-113,
1990[Medline].
3.
Becq, F.,
M. D. Merten,
M. A. Voelckel,
M. Gola,
and
C. Figarella.
Characterization of cAMP dependent CFTR-chloride channels in human tracheal gland cells.
FEBS Lett.
321:
73-78,
1993[Medline].
4.
Berger, H. A.,
S. M. Travis,
and
M. J. Welsh.
Regulation of the cystic fibrosis transmembrane conductance regulator Cl
channel by specific protein kinases and protein phosphatases.
J. Biol. Chem.
268:
2037-2047,
1993[Abstract/Free Full Text].
5.
Berschneider, H. M.,
M. R. Knowles,
R. G. Azizkhan,
R. C. Boucher,
N. A. Tobey,
R. C. Orlando,
and
D. W. Powell.
Altered intestinal chloride transport in cystic fibrosis.
FASEB J.
2:
2625-2629,
1988[Abstract/Free Full Text].
6.
Brayden, D. J.,
M. R. Hanley,
O. Thastrup,
and
A. W. Cuthbert.
Thapsigargin, a new calcium-dependent epithelial anion secretagogue.
Br. J. Pharmacol.
98:
809-816,
1989[Abstract].
7.
Brayden, D. J.,
M. E. Krouse,
T. Law,
and
J. J. Wine.
Stilbenes stimulate T84 Cl
secretion by elevating Ca2+.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G325-G333,
1993[Abstract/Free Full Text].
8.
Clarke, L. L.,
B. R. Grubb,
J. R. Yankaskas,
C. U. Cotton,
A. McKenzie,
and
R. C. Boucher.
Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(
/
) mice.
Proc. Natl. Acad. Sci. USA
91:
479-483,
1994[Abstract].
9.
Engelhardt, J. F.,
J. R. Yankaskas,
S. A. Ernst,
Y. Yang,
C. R. Marino,
R. C. Boucher,
J. A. Cohn,
and
J. M. Wilson.
Submucosal glands are the predominant site of CFTR expression in the human bronchus.
Nat. Genet.
2:
240-248,
1992[Medline].
10.
Engelhardt, J. F.,
M. Zepeda,
J. A. Cohn,
J. R. Yankaskas,
and
J. M. Wilson.
Expression of the cystic fibrosis gene in adult human lung.
J. Clin. Invest.
93:
737-749,
1994[Medline].
11.
Finkbeiner, W. E.,
S. D. Carrier,
and
C. E. Teresi.
Reverse transcription-polymerase chain reaction (RT-PCR) phenotypic analysis of cell cultures of human tracheal epithelium, tracheobronchial glands, and lung carcinomas.
Am. J. Respir. Cell Mol. Biol.
9:
547-556,
1993[Medline].
12.
Fischer, H.,
K. M. Kreusel,
B. Illek,
T. E. Machen,
U. Hegel,
and
W. Clauss.
The outwardly rectifying Cl
channel is not involved in cAMP-mediated Cl
secretion in HT-29 cells: evidence for a very-low-conductance Cl
channel.
Pflügers Arch.
422:
159-167,
1992[Medline].
13.
Foskett, J. K.,
and
D. C. Wong.
[Ca2+]i inhibition of Ca2+ release-activated Ca2+ influx underlies agonist- and thapsigargin-induced [Ca2+]i oscillations in salivary acinar cells.
J. Biol. Chem.
269:
31525-31532,
1994[Abstract/Free Full Text].
14.
Haas, M.,
D. G. McBrayer,
and
J. R. Yankaskas.
Dual mechanisms for Na-K-Cl cotransport regulation in airway epithelial cells.
Am. J. Physiol.
264 (Cell Physiol. 33):
C189-C200,
1993[Abstract/Free Full Text].
15.
Haws, C.,
W. E. Finkbeiner,
J. H. Widdicombe,
and
J. J. Wine.
CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl
conductance.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L502-L512,
1994[Abstract/Free Full Text].
16.
Jiang, C.,
W. E. Finkbeiner,
J. H. Widdicombe,
and
S. S. Miller.
Fluid transport across cultures of human tracheal glands is altered in cystic fibrosis.
J. Physiol. (Lond.)
501:
637-647,
1997[Abstract].
17.
Kachintorn, U.,
M. Vajanaphanich,
A. E. Traynor-Kaplan,
K. Dharmsathaphorn,
and
K. E. Barrett.
Activation by calcium alone of chloride secretion in T84 epithelial cells.
Br. J. Pharmacol.
109:
510-517,
1993[Abstract].
18.
Kreusel, K. M.,
M. Fromm,
J. D. Schulzke,
and
U. Hegel.
Cl
secretion in epithelial monolayers of mucus-forming human colon cells (HT-29/B6).
Am. J. Physiol.
261 (Cell Physiol. 30):
C574-C582,
1991[Abstract/Free Full Text].
19.
Lin, W. W.,
and
D. M. Chuang.
Different signal transduction pathways are coupled to the nucleotide receptor and the P2Y receptor in C6 glioma cells.
J. Pharmacol. Exp. Ther.
269:
926-931,
1994[Abstract].
20.
McEwan, G. T.,
B. H. Hirst,
and
N. L. Simmons.
Carbachol stimulates Cl
secretion via activation of two distinct apical Cl
pathways in cultured human T84 intestinal epithelial monolayers.
Biochim. Biophys. Acta
1220:
241-247,
1994[Medline].
21.
Nagaki, M.,
S. Shimura,
T. Irokawa,
T. Sasaki,
T. Oshiro,
M. Nara,
Y. Kakuta,
and
K. Shirato.
Bradykinin regulation of airway submucosal gland secretion: role of bradykinin receptor subtype.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L907-L913,
1996[Abstract/Free Full Text].
22.
Sato, K.,
and
F. Sato.
Defective beta adrenergic response of cystic fibrosis sweat glands in vivo and in vitro.
J. Clin. Invest.
73:
1763-1771,
1984[Medline].
23.
Shen, B. Q.,
W. E. Finkbeiner,
J. J. Wine,
R. J. Mrsny,
and
J. H. Widdicombe.
Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl
secretion.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L493-L501,
1994[Abstract/Free Full Text].
24.
Shen, B. Q.,
R. J. Mrsny,
W. E. Finkbeiner,
and
J. H. Widdicombe.
Role of CFTR in chloride secretion across human tracheal epithelium.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L561-L566,
1995[Abstract/Free Full Text].
25.
Singh, M.,
M. Krouse,
S. Moon,
and
J. J. Wine.
Most basal Isc in Calu-3 human airway cells is bicarbonate-dependent Cl
secretion.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L690-L698,
1997[Abstract/Free Full Text].
26.
Solc, C. K.,
and
J. J. Wine.
Swelling-induced and depolarization-induced Cl
channels in normal and cystic fibrosis epithelial cells.
Am. J. Physiol.
261 (Cell Physiol. 30):
C658-C674,
1991[Abstract/Free Full Text]. 31): January 1992, following table of contents.]
27.
Stutts, M. J.
Regulation of other airway epithelial ion channels by CFTR.
Cystic Fibrosis
Current Topics
3:
91-106,
1996.
28.
Tabcharani, J. A.,
A. Boucher,
J. W. Eng,
and
J. W. Hanrahan.
Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium, nucleotides and kinases.
J. Membr. Biol.
142:
255-266,
1994[Medline].
29.
Tabcharani, J. A.,
X. B. Chang,
J. R. Riordan,
and
J. W. Hanrahan.
Phosphorylation-regulated Cl
channel in CHO cells stably expressing the cystic fibrosis gene.
Nature
352:
628-631,
1991[Medline].
30.
Tabcharani, J. A.,
W. Low,
D. Elie,
and
J. W. Hanrahan.
Low-conductance chloride channel activated by cAMP in the epithelial cell line T84.
FEBS Lett.
270:
157-164,
1990[Medline].
31.
Thastrup, O.,
P. J. Cullen,
B. K. Drobak,
M. R. Hanley,
and
A. P. Dawson.
Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase.
Proc. Natl. Acad. Sci. USA
87:
2466-2470,
1990[Abstract].
32.
Veeze, H. J.,
D. J. Halley,
J. Bijman,
J. C. de Jongste,
H. R. de Jonge,
and
M. Sinaasappel.
Determinants of mild clinical symptoms in cystic fibrosis patients. Residual chloride secretion measured in rectal biopsies in relation to the genotype.
J. Clin. Invest.
93:
461-466,
1994[Medline].
33.
Weymer, A.,
P. Huott,
W. Liu,
J. A. McRoberts,
and
K. Dharmsathaphorn.
Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line.
J. Clin. Invest.
76:
1828-1836,
1985[Medline].
34.
Widdicombe, J. H.
Altered regulation of airway fluid content in cystic fibrosis.
Cystic Fibrosis
Current Topics
2:
109-129,
1994.
35.
Yamaya, M.,
W. E. Finkbeiner,
and
J. H. Widdicombe.
Altered ion transport by tracheal glands in cystic fibrosis.
Am. J. Physiol.
261 (Lung Cell. Mol. Physiol. 5):
L491-L494,
1991[Abstract/Free Full Text].
36.
Yamaya, M.,
W. E. Finkbeiner,
and
J. H. Widdicombe.
Ion transport by cultures of human tracheobronchial submucosal glands.
Am. J. Physiol.
261 (Lung Cell. Mol. Physiol. 5):
L485-L490,
1991[Abstract/Free Full Text].
37.
Yamaya, M.,
T. Ohrui,
W. E. Finkbeiner,
and
J. H. Widdicombe.
Calcium-dependent chloride secretion across cultures of human tracheal surface epithelium and glands.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L170-L177,
1993[Abstract/Free Full Text].
38.
Yamaya, M.,
K. Sekizawa,
Y. Kakuta,
T. Ohrui,
T. Sawai,
and
H. Sasaki.
P2u-purinoceptor regulation of chloride secretion in cultured human tracheal submucosal glands.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L979-L984,
1996[Abstract/Free Full Text].
AJP Lung Cell Mol Physiol 273(6):L1208-L1219