1 Cystic Fibrosis Research
Laboratory, The Calu-3 cell
line is being investigated as a model for human submucosal gland serous
cells. In a previous investigation of basal short-circuit current
(Isc) in Calu-3
cells, high levels of bumetanide-insensitive basal
Isc (~60
µA/cm2) were measured in cells
grown at an air interface. Basal
Isc was reduced
only 7% by bumetanide, and the largest component of basal
Isc required both
Cl
cystic fibrosis; Ussing chamber; epithelia; submucosal gland; cell
culture
CYSTIC FIBROSIS (CF) arises from mutations in the CF
transmembrane conductance regulator (CFTR), an anion channel located primarily in the apical membranes of epithelia. Lung disease is the
most life-threatening consequence of CF, but it remains unclear exactly
how defects in CFTR lead to lung infections. Submucosal gland serous
cells are the predominant sites of CFTR expression in the human lung
(4), so it seems important to understand the basis of ion transport in
these cells. The Calu-3 cell line is being evaluated as a model for ion
transport by human airway serous cells (5, 14, 17,
18).
An appreciable basal short-circuit current
(Isc) is
present in unstimulated Calu-3 cells (14, 17, 18). In previous work, the basal Isc was
shown to be completely insensitive to apical amiloride (17, 18) and to
be inhibited only slightly by bumetanide (18). Various blockers and
ion-substitution experiments were used to identify three
bumetanide-insensitive transport processes that account for most of the
basal Isc.
Phlorizin inhibits ~20% of the
Isc by blocking
Na+/glucose cotransport (18).
Removing Those results provided clear evidence for the importance of
The results of Singh et al. (18) are compatible with either
We have now tested this model by measuring transepithelial fluxes of
36Cl The Calu-3 cell line was obtained and grown as in previous studies (14,
18). Cells were passaged with a 1:4 dilution or plated at
106
cells/cm2 onto human placental
collagen-coated Costar Snapwell inserts (0.45-µm pore size,
1.13-cm2 surface area; Costar,
Cambridge, MA). Cells grew on inserts at least 10 days before use. In
contrast to previous studies, the filters were not cut from the inserts
but were instead mounted in chambers that accommodated the inserts,
thus avoiding edge damage. The medium was changed every 2-3 days
with an air interface culturing in which medium was added only to the
basolateral side of the inserts.
Commercially available Ussing chambers (World Precision Instruments,
Sarasota, FL) were used to mount the Snapwell filters without edge
damage. Both sides of the monolayers were bathed with 10 ml of
bicarbonate-buffered (25 mM) Krebs-Henseleit solution (300 mosmol/l, pH
7.4 at 37°C when gassed with 95%
O2-5%
CO2). Drugs were added in small
volumes from concentrated stock solutions. The transepithelial
conductance was estimated at 2-s intervals by measuring responses to 1- or 2-mV amplitude pulses. Because oscillations in
Isc were
sometimes present,
Isc was
determined by measuring the area under the curve of
Isc vs. time. Positive values represent anion
secretion or Na+ absorption.
For flux measurements, 5 or 10 µCi of
36Cl All chemicals were reagent grade and, unless otherwise specified,
obtained from Sigma (St. Louis, MO). Stock solutions of forskolin
(Calbiochem, La Jolla, CA) in ethanol and thapsigargin in dimethyl
sulfoxide were stored at Statistics. Tissue pairs were matched
within 0.5 mS/cm2 of baseline transepithelial conductance.
Data are reported as means ± SE. Statistical significance was
assessed with two-tailed Student's t-test.
P values < 0.05 were regarded as
statistically significant.
Measurement of
36Cl
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
and
in the bathing solutions. Because Isc could be
partially inhibited by basolateral
4,4'-dinitrostilbene-2,2'-disulfonic acid and because the
only known apical exit pathway for anions is the cystic fibrosis
transmembrane conductance regulator, which has a relatively poor
conductance for
, it was concluded
that most basal
Isc is
-dependent Cl
secretion [M.
Singh, M. Krouse, S. Moon, and J. J. Wine. Am. J. Physiol. 272 (Lung Cell. Mol.
Physiol. 16): L690-L698, 1997]. We have now
measured isotopic fluxes of
36Cl
and
22Na+
across short-circuited Calu-3 cells and found that virtually none of
the basal Isc is
Cl
secretion or
Na+ absorption. Thus, in contrast
to the earlier report, we conclude that the major component of basal
Isc is
secretion. Stimulation recruits
primarily Cl
secretion, as
previously proposed.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
and CO2 from the bath eliminates most
of the remaining
Isc, whereas removing Cl
eliminates all
phlorizin-insensitive basal
Isc. Basal
Isc was also
reduced by acetazolamide, which slows the production of
by inhibiting carbonic anhydrase,
and by high levels of basolateral 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), which
inhibits Cl
/
exchange (18).
to basal
Isc, but what
role does
play? Because virtually
all Isc except
that inhibited by phlorizin was lost when
Cl
was removed from the
medium (18), Cl
-independent
secretion (19, 21) was excluded.
-dependent
Cl
secretion or
Cl
-dependent
secretion. However, several findings led Singh et al. to favor
-dependent Cl
secretion. First, apical
Cl
/
exchange seemed absent because there was no inhibition of
Isc with very
high levels of apical DNDS or
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Second,
inhibition by basolateral DNDS indicated the apparent presence of a
basolateral
Cl
/
exchanger. Third, CFTR has a relatively low conductance to
(15). In the proposed model (18),
basolateral
Cl
/
exchange is used to concentrate intracellular Cl
concentration above the
Cl
equilibrium
potential (6, 13, 20, 22).
and
22Na+.
Contrary to the model, no net serosal-to-mucosal
Cl
flux was detected during
15- to 30-min periods of basal
Isc that averaged
46 ± 1 µA/cm2. In addition,
no net Na+ absorption was
detected. Because most basal
Isc is eliminated by removal of either Cl
or
from the medium, we conclude, in
contrast to the earlier report (18), that the majority of basal
Isc is Cl
-dependent
secretion.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
or
22Na+
were placed on either the mucosal or serosal side. One- to
three-milliliter aliquots were removed from the cold side at 5- to
10-min intervals and replaced with fresh medium. Fifty-microliter
aliquots were removed from the hot side at least every 30 min. To
measure
36Cl
,
three times the sample volume of scintillation fluid was added, and the
mixture was counted in a Beckman LS6000 SC liquid scintillation counter. Samples containing
22Na+
were measured in a Packard 1500LS gamma counter. Net flux was determined as the difference between fluxes in the mucosal-to-serosal and serosal-to-mucosal directions.
20°C. Stock solutions of bumetanide, phlorizin, and acetazolamide in dimethyl sulfoxide were
stored at 4°C.
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
fluxes during basal and stimulated
Isc.
In six matched pairs of tissues (mean transepithelial resistance = 232 ± 38
· cm2),
isotopic Cl
fluxes were
measured for periods of 15-30 min during which basal (unstimulated)
Isc averaged 46 ± 1 µA/cm2. As shown in Fig.
1A
and Table 1, no significant net flux of 36Cl
was observed during this period. Apical addition of 200 µM phlorizin decreased Isc by
12 ± 1 µA/cm2 but produced
no significant change in Cl
flux. Bilateral application of 10 µM forskolin caused little change
in either Isc or
net Cl
flux. Subsequent
addition of 300 nM thapsigargin increased
Isc to 157 ± 20 µA/cm2 and net
serosal-to-mucosal Cl
flux
to 3.06 ± 0.47 µeq · cm
2 · h
1
(
82 ± 12 µA/cm2;
n = 3 tissues). Thus, in each
condition, a large portion of the
Isc was not
accounted for by Cl
secretion. This residual
Isc, which may
represent some combination of
secretion and Na+ absorption,
accounted for virtually all of the basal
Isc and approximately one-third of the
Isc stimulated by
thapsigargin.
View larger version (24K):
[in a new window]
Fig. 1.
Comparison of short-circuit current
(Isc) and
isotopic fluxes. A:
Cl fluxes.
B:
Na+ fluxes. Measured
Isc, mean
Isc for condition
indicated; Cl
Isc Equivalent
and Na+
Isc Equivalent,
equivalent Isc
calculated for
36Cl
and
22Na+,
respectively; Residual
Isc, difference
between the two. Secretion of
36Cl
was only observed after treatment with thapsigargin (Tg), which in
these experiments was always added after forskolin (Fsk).
Table 1.
Basal and stimulated Na+ and Cl
fluxes and corresponding Isc
Measurement of
22Na+
fluxes during basal and stimulated
Isc.
To determine the possible contribution of
Na+ absorption to basal
Isc, isotopic
Na+ fluxes were measured in four
matched pairs of tissues (mean transepithelial resistance = 408 ± 34 · cm2)
for periods of 15-30 min during which basal (unstimulated)
Isc averaged 42 ± 5 µA/cm2. The net
serosal-to-mucosal flux of
22Na+
was 0.29 ± 0.14 µeq · cm
2 · h
1
(equivalent Isc
of
8 ± 4 µA/cm2),
which is not significantly different from zero and indicates that the
basal Isc cannot
be accounted for by Na+
absorption. (Basal
Isc was lower
than usual in this batch of filters, and phlorizin caused no change in
Isc, suggesting
that the Na+/glucose cotransporter
was inoperative in these filters.) In two matched tissues, application
of forskolin+thapsigargin caused Isc to increase
to 104 µA/cm2, with no
significant change in the flux of
22Na+
(Fig. 1B, Table 1).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Past work (14, 17, 18) has shown that Calu-3 cells generate large basal
and stimulated
Isc values. The
majority of stimulated Isc was shown to
be Cl secretion by flux
measurements (17), but the ionic basis for basal
Isc was not
established. Singh et al. (18) proposed that most basal
Isc was
-dependent
Cl
secretion, based
primarily on the abolition of all basal
Isc (except the
phlorizin-sensitive component) after the removal of Cl
and the ability of
basolateral but not apical DNDS to inhibit basal
Isc. The present
results negate that model and, instead, support a model in which most
basal Isc is
secretion, whereas
most stimulated
Isc is
Cl
secretion (Fig.
2). This model is consistent with models
for
secretion in the duodenum and
epididymus of rodents, (2, 8, 10), although in the latter tissues, an
Na+/
cotransporter may also play a role (2).
|
The model shown in Fig. 2 is consistent with many, but not all, of our
observations. It is consistent with
and
CO2 dependence of basal
Isc, with an
insensitivity of basal Isc to
bumetanide, with a reduction in basal
Isc by
acetazolamide, and with a reduction in basal
Isc by
5-(N-ethyl-N-isopropyl)-amiloride (Penland, unpublished observations). However, it does not
account for the elimination of basal
Isc in
Cl
-free medium. (It was
this observation that originally suggested that basal
Isc might be an
-dependent
Cl
secretion.) At
present, we have no experimental data that can explain the
Cl
dependence of basal
Isc. However, if
the model is correct as shown, then removal of
Cl
must either open an
equivalent basolateral path for
exit, reduce the production of
to
a level that is in electrochemical equilibrium with external
, or cause CFTR to close.
[There is precedent for regulation of Cl
-channel conductance by
cytosolic anions in some cells (3).] The model also does not
explain the inhibitory effect of DNDS on basal
Isc. The model of
Singh et al. (18) proposed that DNDS was inhibiting a basolateral anion
exchanger, but such an exchanger is not compatible with the present
model. A possibility consistent with the present model is that DNDS
inhibits a basolateral
Na+/
cotransporter that elevates intracellular
concentration.
Does exit the apical membrane
exclusively via CFTR? Single-channel recordings (9) and measurements of
Isc through
basolaterally permeabilized Calu-3 monolayers (14) suggest that CFTR is
the major and possibly only physiologically relevant anion channel in
the apical membrane. Although
conductance through CFTR has been controversial, a variety of measurements indicate that CFTR has a low but significant conductance to
(7, 11, 16). In Calu-3 apical membranes specifically, Illek et al. (11) have shown that the average
relative permselectivity for
vs. Cl
is ~15%. Thus at
least some
could exit via CFTR,
but the exact role of CFTR in
secretion remains to be determined. Our measurements of
Na+ fluxes, although sufficient to
eliminate Na+ absorption as the
basis for basal
Isc, were not
sufficient to rule out a contribution of
Na+/
cotransport (2) to basal
Isc.
Speculation on functional consequences of CFTR
mutations. In the human genetic disease CF, conductance
through CFTR is lost. If the CFTR is the exclusive exit pathway for
in Calu-3 cells and if Calu-3
cells mimic serous cells, loss of CFTR conductance will eliminate all
basal and stimulated ion and fluid secretion, leaving only
Na+/glucose absorption. Under
these conditions, secreted macromolecules would be undiluted and
trapped within the glands where unopposed absorption would desiccate
them further. In addition,
contributed by the glands would be absent from airway surface liquid,
and the pH of the secreted fluid might be more acidic. An analogous
condition has been simulated by pharmacologically blocking both
Cl
transport and
transport in bronchial submucosal glands from the pig, with the result that mucins accumulated in the
glands (12).
Because of their abundant production of bactericidal compounds, serous
cells are considered to be "the primary defensive cell of the
mucosa" (1). Reduced fluid secretion by serous cells should reduce
the dissemination of antibiotics in surface fluid and could contribute
to the reduced mucosal defenses that are characteristic of CF.
Therefore, it is of considerable interest to determine whether, in
fact, all secretion in serous
cells requires CFTR or whether alternate transporters exist in parallel
with CFTR.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Heart, Lung, and Blood Institute Grant HL-42368 (to J. H. Widdicombe).
![]() |
FOOTNOTES |
---|
M. C. Lee was the recipient of a Howard Hughes Summer Research Fellowship from the Stanford University Department of Biological Sciences. C. M. Penland is a postdoctoral fellow of the Cystic Fibrosis Foundation.
Address for reprint requests: J. J. Wine, Cystic Fibrosis Research Laboratory, Bldg. 420 (Jordan Hall), Stanford Univ., Stanford, CA 94305-2130.
Received 23 October 1997; accepted in final form 9 December 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Basbaum, C. B.,
B. Jany,
and
W. E. Finkbeiner.
The serous cell.
Annu. Rev. Physiol.
52:
97-113,
1990[Medline].
2.
Chan, H. C.,
W. H. Ko,
W. Zhao,
W. O. Fu,
and
P. Y. Wong.
Evidence for independent Cl and
secretion and involvement of an apical Na(+)-
cotransporter in cultured rat epididymal epithelia.
Exp. Physiol.
81:
515-524,
1996[Abstract].
3.
Dinudom, A.,
J. A. Young,
and
D. I. Cook.
Na+ and Cl conductances are controlled by cytosolic Cl
concentration in the intralobular duct cells of mouse mandibular glands.
J. Membr. Biol.
135:
289-295,
1993[Medline].
4.
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].
5.
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].
6.
Forte, J. G.,
and
W. W. Reenstra.
Chloride transport by gastric mucosa.
In: Advances in Comparative and Environmental Physiology. Berlin: Springer-Verlag, 1994, p. 239-259.
7.
Gray, M. A.,
S. Plant,
and
B. E. Argent.
cAMP-regulated whole cell chloride currents in pancreatic duct cells.
Am. J. Physiol.
264 (Cell Physiol. 31):
C591-C602,
1993
8.
Guba, M.,
M. Kuhn,
W. G. Forssmann,
M. Classen,
M. Gregor,
and
U. Seidler.
Guanylin strongly stimulates rat duodenal secretion: proposed mechanism and comparison with other secretagogues.
Gastroenterology
111:
1558-1568,
1996[Medline].
9.
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
10.
Hogan, D. L.,
D. L. Crombie,
J. I. Isenberg,
P. Svendsen,
O. B. Schaffalitzky de Muckadell,
and
M. A. Ainsworth.
CFTR mediates cAMP- and Ca2+-activated duodenal epithelial secretion.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G872-G878,
1997
11.
Illek, B.,
J. R. Yankaskas,
and
T. E. Machen.
cAMP and genistein stimulate conductance through CFTR in human airway epithelia.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L752-L761,
1997
12.
Inglis, S. K.,
M. R. Corboz,
A. E. Taylor,
and
S. T. Ballard.
Effect of anion transport inhibition on mucus secretion by airway submucosal glands.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L372-L377,
1997
13.
Kizer, N. L.,
D. Vandorpe,
B. Lewis,
B. Bunting,
J. Russell,
and
B. A. Stanton.
Vasopressin and cAMP stimulate electrogenic chloride secretion in an IMCD cell line.
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F854-F861,
1995
14.
Moon, S.,
M. Singh,
M. Krouse,
and
J. J. Wine.
Calcium-stimulated Cl secretion in Calu-3 human airway cells requires CFTR.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L1208-L1219,
1997
15.
Poulsen, J. H.,
H. Fischer,
B. Illek,
and
T. E. Machen.
Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator.
Proc. Natl. Acad. Sci. USA
91:
5340-5344,
1994[Abstract].
16.
Poulsen, J. H.,
and
T. E. Machen.
-dependent pHi regulation in tracheal epithelial cells.
Pflügers Arch.
432:
546-554,
1996[Medline].
17.
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
18.
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
19.
Smith, J. J.,
and
M. J. Welsh.
cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia.
J. Clin. Invest.
89:
1148-1153,
1992[Medline].
20.
Tessier, G. J.,
T. R. Traynor,
M. S. Kannan,
and
S. M. O'Grady.
Mechanisms of sodium and chloride transport across equine tracheal epithelium.
Am. J. Physiol.
259 (Lung Cell. Mol. Physiol. 3):
L459-L467,
1990
21.
Van Scott, M. R.,
C. M. Penland,
C. A. Welch,
and
E. Lazarowski.
Beta-adrenergic regulation of Cl and
secretion by Clara cells.
Am. J. Respir. Cell Mol. Biol.
13:
344-351,
1995[Abstract].
22.
Vaughan-Jones, R. D.
An investigation of chloride-bicarbonate exchange in the sheep cardiac Purkinje fibre.
J. Physiol. (Lond.)
379:
377-406,
1986[Abstract].