Effects of Endothelin-1 on Epithelial Ion Transport in Human Airways
Sabine Blouquit,* Anouar Sari,* Alain Lombet, Michelle D’herbomez, Emmanuel Naline, Regis Matran,
and Thierry Chinet
Laboratoire de Biologie et Pharmacologie des Epithe ´liums Respiratoires, Boulogne; INSERM U339 UFR Saint Antoine,
Paris; De ´partement de Biophysique, University of Lille, Lille; Institut de Pharmacologie, Paris, France; and De ´partement
de Physiologie, University of Lille, Lille, France
Endothelin-1 (ET-1) exerts many biological effects in airways,
including bronchoconstriction, airway mucus secretion, cell
proliferation, and inflammation. We investigated the effect of
ET-1 on Na absorption and Cl secretion in human bronchial
epithelial cells. Addition of 10?7M ET-1 had no effect on the
inhibition of the short circuit current (Isc) induced by amiloride,
a Na channel blocker. Addition of 10?7M ET-1 to the apical bath
in the presence of amiloride increased Isc in cultured human
bronchial epithelial cells studied in Ussing chambers. No effect
ing that the involved ET-1 receptors are likely present only in
the apical membrane of the cells. Use of Cl-free solutions and
bumetanide reduced the ET-1–induced increases in Isc, indicat-
ing that ET-1 stimulates Cl secretion. The ET-1–induced increase
in Isc was prevented by exposure to the ETBreceptor antagonist
BQ-788 but not to the ETAreceptor antagonist BQ-123. ET-1 did
not raise intracellular Ca levels, but increased the intracellular
concentration of cAMP. These findings indicate that ET-1 is a
apically located ETB receptors and activation of the cAMP
Endothelin 1 (ET-1) belongs to a family of potent peptidic
vasoconstrictor agents that exert an array of biological ef-
fects in addition to constriction of the vascular smooth mus-
cle cells (1, 2). In airways, several studies have shown that
chial smooth muscle cells. ET-1 also acts on airway epithe-
lial cells. Animal studies have found that ET-1 increases
the cilia beat frequency, activates mucus secretion by sub-
mucosal glands, and stimulates the proliferation of epithe-
lial cells (3–7). In humans, ET-1 stimulates lactoferrin and
mucous glycoprotein release from serous and mucous cells
in cultured nasal mucosal explants (8), and may affect ex-
pression of genes in bronchial epithelial cells such as the
fibronectin gene (9). The presence of immunoreactive ET-1
(Received in original form July 5, 2002 and in revised form January 29,
*These authors contributed equally to the work presented in this article.
Address correspondence to: Professeur Thierry Chinet, Laboratoire de Bio-
logie et Pharmacologie des Epithe ´liums Respiratoires, UFR Paris Ile de
France Ouest, Universite ´ de Versailles Saint Quentin en Yvelines, Ho ˆpital
Abbreviations: intracellular free Ca concentration, [Ca]i; cyclic adenosine
monophosphate, cAMP; Dulbecco’s modified Eagle’s medium and Ham’s
F12 mixture, DMEM/F12; endothelin-1, ET-1; short circuit current, Isc;
Am. J. Respir. Cell Mol. Biol.Vol. 29, pp. 245–251, 2003
Originally Published in Press as DOI: 10.1165/rcmb.2002-0104OC on March 6, 2003
Internet address: www.atsjournals.org
and abundant binding sites for ET-1 in airways suggests
that this peptide is an important autocrine and/or paracrine
neuromodulator of airway functions (10, 11).
In view of these data, we raised the hypothesis that ET-1
may also contribute to the regulation of transepithelial ion
transport, another major function of human airway epithe-
lial cells. Airway epithelial ion transport processes regulate
the volume of airway surface liquid and airway secretions
(12). The net movement of salt and fluid across human
airway epithelium is generally regarded as the result of
two opposite active ion transports: Na absorption and Cl
secretion. Active Na absorption predominates in the basal
state and induces fluid absorption from the lumen. Active
Cl secretion is the driving force for fluid secretion in human
airways and can be stimulated by various agents, including
adenosine 3?,5?-cyclic monophosphate (cAMP)-activating
agents and purinergic agonistssuch as adenosine 5?-triphos-
ET-1 could affect Na absorption across human airway
epithelium because recently published data suggest that
ET-1 may be an important negative regulator of ENaC:
adult rats lacking functional ETBreceptor activity display
enhanced Na absorption in the distal nephron (13); further-
more, in vitro studies in cell lines expressing ENaC have
demonstrated that ET-1 potently inhibits ENaC via ETB
receptors, and that this effect is mediated by Src family
ET-1 could also participate in the regulation of Cl secre-
tion across human airway epithelium because intranasal
administration of ET-1 in allergic and nonallergic subjects
induces symptoms of rhinorrhea and increases the amount
of secretions (15) and because this peptide regulates Cl
secretion in other epithelia. However, the effects of ET-1
on epithelial Cl secretion–i.e., the nature of the effect (stim-
differ between tissues and between species. In human gall-
bladder, ET-1 inhibits cAMP-induced Cl secretion (16),
whereas in human intestine, ET-1 stimulates Cl secretion
in part via the activation of enteric nerves (17). In dog
airways, ET-1 increases electrogenic Cl secretion (3, 18,
of cyclooxygenase products such as PGE2.
The goal of this study was therefore to determine
whether ET-1 regulates active Na absorption and/or active
Cl secretion in human airway epithelium, and to describe
the mechanisms of ET-1–mediated regulation of transepi-
thelial ion transport. We used cultured human bronchial
epithelial cells to ensure that the effect of ET-1 on ion
transport would not be mediated by nonepithelial airway
mucosal cells and by airway nerves.
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 29 2003
Materials and Methods
Human bronchi were obtained from pieces of lobectomy or pneu-
(age 61.7 ? 2.6 yr; 21 male, all smokers) in agreement with the
current French legislation. Tissues used for experimental studies
were taken from macroscopically normal areas distant from the
pathological tissue. Bronchi were rinsed with Ham’s F12 medium
(Sigma Chemicals, Saint Quentin Fallavier, France), dissected free
of adjacent parenchyma, washed again with F12, and incubated
for 2–12 h at 4?C in Dulbecco’s modified Eagle’s medium (Gibco,
Cergy Pontoise, France) and Ham’s F12 (1:1) mixture (DMEM/
F12) supplemented with 100 U/ml penicillin, 100 ?g/ml streptomy-
cin,50 ?g/mlgentamycin, and5 ?g/mlfungizone. Thepreparations
were then washed with DMEM/F12 and incubated in DMEM/F12
with 0.1% protease and 0.01% DNase for 48 h at 4?C. Fetal bovine
serum (10%) was then added to neutralize the enzymes, and cells
were detached by gentle agitation. The resulting suspension was
centrifuged (500 ? g, 5 min, 10?C). The cell pellet was resuspended
and plated on home-made collagen matrix supports affixed to an
orifice drilled in polycarbonate cups at a density of 5 ? 105cells/
cm2.Thediameter oftheaperturewas3.5mm. Thecollagenmatrix
was made of calfskin type I collagen, which was diluted in 0.2%
acetic acid at a concentration of 15 mg/ml, mixed 2:1 with glutaral-
dehyde (2.5% in phosphate-buffered saline [PBS]) and spread
over the orifice of the polycarbonate cups. The culture medium
consisted of Ham’s F12 supplemented with: insulin (5 ?g/ml),
endothelial cell growth supplement (2 ?g/ml), epithelial growth
factor (25 ng/ml), hydrocortisone (10?6M), transferrin (7.5 ?g/ml),
streptomycin (100 ?g/ml), gentamycin (50 ?g/ml), and amphoteri-
cin B (5 ?g/ml). The preparations were fed every other day with
culture medium and incubated at 37?C, 5% CO2–95% air, in a
tissue culture incubator.
Transepithelial Electrical Recordings
Five to ten days after plating, the cell culture preparations were
mounted in Ussing-type chambers. The apical and basolateral sur-
faces of the preparations were bathed by 10 ml of Kreb’s bicarbon-
ate Ringer (KBR) solutions gassed with 95% O2–5% CO2. Experi-
ments were conducted at 37?C. The short-circuit current (Isc) was
monitored continuously using a DVC1000 voltage clamp (WPI,
5–10 min. Voltage-sensing electrodes consisted of 3 mol/liter KCl-
agar bridges; the reference electrode was placed at the basolateral
side. Current-passing bridges consisted of KBR-agar bridges.
Transepithelial resistance (R) was determined by clamping the PD
at ?10 mV at 25-s intervals, recording the deflection in Isc, and
applying Ohm’s law. Cell preparations were allowed to equilibrate
until stabilization of bioelectric variables which required ? 20–40
min. Basal bioelectric activity was monitored for 10 min before
addition of drugs.
The following pharmaceutical agents were used: ET-1, amilor-
ide, forskolin, ATP, the ETAreceptor-selective antagonist BQ123
and the ETBreceptor-selective antagonist BQ788. Agents were
added to the apical and/or basolateral bathing solutions, and bio-
ide (10?5M), an Na channel inhibitor, was added first to inhibit
Na absorption. The amiloride-sensitive Isc is a measure of Na
absorption, and the residual amiloride-insensitive Isc is a measure
of Cl secretion (12). The Cl secretagogues forskolin and ATP were
added sequentially in the presence of amiloride. Forskolin (10?5
M), an activator of the cAMP pathway, was added to the apical
and basolateral baths, whereas ATP (10?4mol/liter), which in-
creases the intracellular Ca concentration, was added to the apical
bath only. ET-1 was added to the apical or the basolateral bath
as indicated. BQ123 (10?6M) and BQ788 (10?6M) were added
to the apical bath only. Changes in R and Isc were calculated as
the variations between the values measured immediately before
the addition of reagents and the values corresponding to the pla-
teau phase after addition of amiloride, ET-1, and forskolin and
corresponding to the maximal change after addition of ATP. To
sides ofthe preparations,and with bumetanide(10?5M),an inhibi-
tor of the Na/K/2Cl cotransporter, in the basolateral solution. To
Cl secretion, we added the Ca chelator 1,2-bis (2-aminophenoxy)-
ethane-N,N,N?,N?-tetraacetic acid acetomethoxy ester (BAPTA-
AM, 10?5M) to the apical and basolateral baths, 60 min before
successive additions of amiloride, ET-1, and ATP.
To determine the role of cyclooxygenase product formation in ET-
ethanol) to the apical and basolateral baths after addition of ami-
loride. The bioelectric variables of the preparations were then
recorded during at least 20 min before addition of ET-1 (10?7M)
to the apical bath.
Measurement of Intracellular cAMP Level
Freshly isolated cultured human bronchial epithelial cells were
grown to confluence on polycarbonate surface coated with colla-
gen. Preparations were exposed for 10 min on their apical side to
ET-1 (10?7M), vehicle (acetic acid 0.2%), or 3-isobutyl-1-methyl-
xanthine (IBMX 10?6M), a cAMP phosphodiesterase inhibitor.
Cells were then lysed by apical addition of 40 ?l of perchloric acid
and centrifuged at 2,350 ? g for 5 min. The excess perchloric acid
in the supernatant was neutralized by potassium carbonate. cAMP
was assayed in the supernatant by radioimmunoassay (Amersham
Pharmacia Biotech, Buckinghamshire, UK). The protein content
of cell suspension was quantified and intracellular cAMP levels
were expressed as pmol/mg protein.
Measurement of Intracellular Free Ca Concentration
To measure intracellular free Ca concentration ([Ca]i), isolated
bronchial epithelial cells were seeded at low density on glass cov-
erslips coated with collagen and fed with culture medium. One or
two days later, they were washed with PBS and loaded with 2.5
?mol/liter Fura-2/AM for 2 h at 37?C in PBS supplemented with
(pH 7.3). After washing with this solution, the coverslips were
placed on an inverted fluorescence microscope connected to a
dynamic imaging system (QuantiCell 700; Visitech International
Ltd, Sunderland, UK). Fluorescence images were recorded every
2 s, and [Ca]iwere calculated from the ratio of the fluorescence
intensities at 340 and 380 nm on a pixel basis. The Ca ionophore
ionomycin wasused to calibratethe Fura-2 fluorescenceratio signal.
The 340 nm/380 nm ratio was converted to an actual [Ca]imeasure-
(20). Foreach preparation, [Ca]iwasmeasured on ?60 cells. After
an initial 1-min long recording, ET-1 (10?7M) or vehicle (acetic
Thapsigargin (10?6M), an inhibitor of the endoplasmic reticulum
Blouquit, Sari, Lombet, et al.: Endothelin-1 and Epithelial Ion Transport in Human Airways 247
Ca2?-ATPase, was then added and fluorescence recorded until a
plateau was reached.
Solutions and Drugs
The composition of the regular KBR solution was (in mmol/liter):
120 NaCl, 0.7 Na2HPO4, 1.5 NaH2PO4, 2 CaCl2, 0.5 MgCl2, 0.45
KCl, 15 NaHCO3, and 1 glucose (pH 7.3). All but 4–6 mM Cl were
replaced with gluconate in the low-Cl solution. The composition
0.15 KH2PO4(pH 7.4). ET-1 was used at a final concentration of
10?9M to 10?6M (stock solution: 10?4M in acetic acid 1N). Fura-2
was obtained from Molecular Probes (Leidin, Netherlands). All
salts and drugs were purchased from Sigma Chemicals, except
BQ123 and BQ788, which were obtained from Bachem (Voisins-
Results are expressed as means ? SEM for n preparations. Com-
parisons were made using the Wilcoxon’s matched pair test and
the unpaired or paired Student’s t test as appropriate. A P ? 0.05
was considered statistically significant.
Transepithelial Electrical Recordings
The baseline PD, Isc, and R of cultured human bronchial
epithelial cells were, respectively, 8.4 ? 1.9 mV, 52.9 ? 8.4
?A/cm2, and 160.5 ? 28.9 ?.cm2(n ? 10). Amiloride in-
then ATP increased Isc by 5.8 ? 1.2 and 32.2 ? 8.2 ?A/
cm2, respectively (n ? 10; P ? 0.05 for both).
eral baths, we observed a significant increase in Isc (2.7 ?
0.5 ?A/cm2, n ? 9, for ET-1, versus 0.2 ? 0.3 ?A/cm2, n ? 7,
for vehicle; P ? 0.05). However, we observed no significant
effect of ET-1 on the decrease in Isc induced by the subse-
quent addition of amiloride (?25.9 ? 7.3 ?A/cm2, n ? 9 for
ET-1, versus –20.4 ? 5.2 ?A/cm2, n ? 7 for vehicle, not
In the presence of amiloride, apical addition of ET-1
(10?7M) was typically characterized by an increase in Isc
followed by a slow decrease toward initial values over ? 10
min (Figure 1). In some instances, a transient decrease in
Isc below initial values followed the initial peak. Addition
of vehicle (acetic acid 0.2%) had no effect on Isc (?Isc ?
?0.5 ? 0.9, n ? 4, NS). Table 1 summarizes the results of
similar experiments. To ascertain that the ET-1–induced
increase in Isc in the presence of amiloride could be attrib-
uted to stimulation of Cl secretion, we performed similar
experiments with low-Cl solutions on both sides and in the
conditions, the ET-1–induced increase in Isc was signifi-
when added to the apical side (Table 1). The responses to
forskolin and ATP were also significantly reduced under
these experimental conditions.
Addition of ET-1 (10?7M) to the basolateral bath of
amiloride-pretreated preparations had no significant effect
Figure 1. Representative tracings of Isc in cultured human bron-
chial epithelial cells exposed to amiloride (10?5M, apical side),
and ATP (10?4M, apical side). Drugs were added sequentially
after stabilization of baseline Isc.
on Isc (?Isc? 0.0 ? 0.3 ?A/cm2, n ? 5, NS). This indicates
that ET-1 stimulates Cl secretion only through receptors
located in the apical side of cultured bronchial epithelial
To construct the dose–effect curve, bronchial epithelial
cell cultures were exposed to concentrations of ET-1 rang-
ing from 10?9M to 10?6M in the presence of amiloride.
The dose–response curve is displayed in Figure 2. The 10?6
M concentration yielded a biphasic response with an in-
crease in Isc immediately followed by a transient decrease
tion (EC50) of ? 10 nM.
Determination of the ET Receptors Mediating
the ET-1–Induced Increase in Cl Secretion
To determine which receptors were involved in the stimula-
tion of Cl secretion by ET-1, the ETAreceptor inhibitor
BQ-123 or the ETBreceptor inhibitor BQ-788 were added
in the apical bath in the presence of amiloride. Neither BQ-
123 (10?6M) nor BQ-788 (10?6M) significantly modified
Isc (?Isc? 0.5 ? 0.4 ?A/cm2and 0.3 ? 0.3 ?A/cm2, respec-
tively; n ? 6 for both). Preparations were exposed to ET-1
15 min later. Pre-incubation with BQ-123 had no effect on
the subsequent ET-1–induced increase in Isc (?Isc? 4.6 ?
1.4 ?A/cm2, n ? 6). In contrast, in the presence of BQ-788,
ET-1 failed to increase Isc (?Isc? ?0.1 ? 0.2 ?A/cm2, n ?
6). These data indicate that ETB, receptors but not ETA
receptors, mediate ET-1–induced Cl secretion.
Effect of Indomethacin on the ET-1–Induced
Increase in Isc
Because previous studies have suggested that stimulation
of epithelial Cl secretion by ET-1 could be attributed to
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 29 2003
Baseline Isc and changes in Isc induced by sequential additions of amiloride, ET-1, forskolin and ATP
in cultured human bronchial epithelial cells
Regular KBR (n ? 9)
Low Cl KBR ? bumetanide (n ? 6)
48.1 ? 5.5
45.9 ? 9.9
?33.7 ? 4.8†
?35.5 ? 8.7†
5.7 ? 1.4†
0.0 ? 0.1*
4.4 ? 1.2†
0.5 ? 0.2*
34.9 ? 10.1†
10.4 ? 4.8†*
Experiments were performed on preparations bathed with regular KBR or with low Cl KBR supplemented with bumetanide (10?5M) in the basolateral bath.
Results are expressed in ?A/cm2(mean ? SE).
*Significantly different (P ? 0.05) as compared with values obtained in regular KBR.
†Significantly different from zero (P ? 0.05).
the effects of indomethacin, an inhibitor of cyclooxygenase
product formation, on the ET-1–induced increase in Isc in
was added to the apical and basolateral baths 20 min before
addition of ET-1 10?7M to the apical bath. As shown in
Table 2, we found no significant effect of indomethacin on
the ET-1–induced increase in Isc.
Effect of ET-1 on cAMP Production
We next tested the effect of ET-1 on the production of
intracellular cAMP in this epithelium. Figure 3 shows that
ET-1 increased cAMP from basal levels of 0.8 ? 0.2 (n ?
5) to 10.3 ? 2.2 pmol/mg protein (n ? 6; P ? 0.01). Apical
addition of IBMX increased cAMP levels to 11.0 ? 1.5
pmol/mg protein (n ? 6; P ? 0.01 compared with control,
NS compared with ET-1).
Effect of ET-1 on [Ca]i
We then studied the effect of ET-1 on [Ca]i. Nine prepara-
tions of cultured human bronchial epithelial cells were
loaded with Fura-2 and exposed to ET-1. Baseline [Ca]i
was 144.2 ? 2.3 nM. No significant change in [Ca]i was
observed after addition of ET-1. For instance, 30 s after
with baseline value). Subsequent addition of thapsigargin
increased [Ca]ito 233.8 ? 14.4 nM (P ? 0.01).
Effect of BAPTA-AM on the ET-1–Induced Increase in Isc
To further investigate the contribution of a Ca second mes-
senger component in the ET-1 effect on Cl secretion, we
Figure 2. ChangesinIscinducedbyvariousconcentrationsofET-1
added in the apical bath of bronchial epithelial cell cultures in the
presence of amiloride (n ? 6). *Significantly different from 10?9
M (P ? 0.05).†Significantly different from 10?7M (P ? 0.05).
exposed the preparations to the Ca chelator BAPTA-AM
for 60 min before addition of amiloride, ET-1, and ATP.
As shown in Table 3, pre-incubation with BAPTA-AM
had no significant effect on basal Isc, amiloride-induced
decrease in Isc, and ET-1–induced increase in Isc. In con-
trast, the ATP-stimulated Cl secretion was significantly in-
hibited by BAPTA.
This study shows that ET-1 has no significant effect on Na
bronchial epithelial cells. ET-1 evoked a sustained increase
in Isc, indicating stimulation of active ion transport. This
effect could be attributed to stimulation of Cl secretion
because ET-1 increased Isc in the presence of amiloride,
an inhibitor of Na absorption, the other major active trans-
epithelial ion transport in airways, and because the increase
in Isc was significantly inhibited by depleting bathing solu-
tions in Cl. In human airways, transepithelial Cl secretion
can be activated by agents that raise the intracellular con-
Ca concentration (12). We herein provide evidence that
ET-1 activates Cl secretion through an increase in cAMP
but not in Ca. The ET-1–induced increase in Cl secretion
was concentration-dependent,and thethreshold concentra-
in the regulation of human airway ion transport.
Previous studies indicate that the effects of ET-1 on
epithelial Cl transport are heterogeneous and vary among
tissues and species in an unpredictable manner. In humans,
Figure 3. Intracellular concentration of cAMP in cultured human
bronchial epithelial cells under baseline conditions and after addi-
tion of ET-1 (10?7M) or IBMX (10?6M) (n ? 6). *Significantly
different from baseline value (P ? 0.01).
Blouquit, Sari, Lombet, et al.: Endothelin-1 and Epithelial Ion Transport in Human Airways 249
Effects of indomethacin on the changes in Isc induced by ET-1 in cultured human bronchial epithelial cells
Indomethacin (n ? 7)Vehicle (n ? 5)
?Isc amiloride (?A/cm2)
?Isc indomethacin or vehicle (?A/cm2)
?Isc ET-1 (?A/cm2)
73.8 ? 10.0
?29.8 ? 8.0*
0.3 ? 0.3
4.8 ? 1.1*
64.8 ? 9.3
?17.3 ? 8.5*
0.0 ? 0.8
6.4 ? 2.5*
Preparations were bathed with regular KBR. After equilibrium, amiloride (10?5M) was added to the apical bath. After stabilization of Isc, indomethacin (10?5M)
or vehicle (ethanol 0.1%) were added in the apical and basolateral baths at least 20 min before exposure to ET-1 (10?7M in the apical bath).
*Significantly different from zero (P ? 0.05). NS: not significant.
for instance, ET-1 inhibits cAMP-dependent anion secre-
tion in gallbladder epithelial cells, but potently stimulates
transepithelial Cl secretion in intestinal epithelial cells (16,
17). The effect of ET-1 on airway epithelial ion transport
had not been studied previously in humans but in animals.
Evidence for ET-1–induced stimulation of transepithelial
Cl secretion was obtained in dog and sheep trachea (3, 18,
19, 21). Our results are consistent with these studies and
suggest that ET-1 acts as a Cl secretagogue in mammalian
airways. The mechanism for this effect may, however, differ
between species. Studies in dog trachea suggest that ET-1
acts through multiple pathways to induce Cl secretion, in-
cluding release of cyclooxygenase products, increase in in-
tracellular Ca levels, and accumulation of cAMP (3, 18,
19). In human airways, we observed that ET-1 increases
intracellular cAMP but not intracellular Ca levels, and that
the ET-1–induced stimulation of Cl secretion is not reduced
by pretreatment with the cyclooxygenase inhibitor indo-
methacin or with the Ca chelator BAPTA-AM.
cell-surface receptors. There are at least two receptor sub-
types, ETAand ETBreceptors. In excised human lungs, one
study found low levels of ETAbut not ETBreceptors in the
bronchial epithelium using a quantitative autoradiographic
technique (22). However, expression of ETBreceptors was
detected in native human bronchial epithelial cells by using
a reverse transcription-polymerase chain reaction–based
ETBreceptors, because the ETBreceptor inhibitor BQ788,
but not the ETAreceptor inhibitor BQ123, prevented the
ET-1–induced stimulation of Cl secretion. In addition, the
Effects of BAPTA-AM on the basal Isc and on the changes in Isc induced by subsequent additions of amiloride,
ET-1, and ATP in cultured human bronchial epithelial cells
BAPTA-AM (n ? 6) Vehicle (n ? 6)
?Isc amiloride (?A/cm2)
?Isc ET-1 (?A/cm2)
?Isc ATP (?A/cm2)
44.5 ? 5.0
?21.1 ? 2.8*
2.1 ? 0.8*
25.3 ? 5.7*
59.8 ? 18.9
?25.5 ? 11.8*
1.9 ? 0.2*
48.7 ? 4.3*
Preparations were bathed with regular KBR and pre-incubated with BAPTA-AM or vehicle for 60 min. Amiloride (10?5M), ET-1 (10?7M), and ATP (10?4M)
were added to the apical bath.
*Significantly different from zero (P ? 0.05); NS: not significant.
EC50in our study was ? 10 nM, which is consistent with
the dissociation constant of ETBreceptor as determined in
other cell types (24). Because ET-1 affected Cl secretion
only when added in the apical bath and not the basolateral
bath, the ETBreceptors that mediate the ET-1–induced Cl
secretion are presumably located in the apical membrane.
Interestingly, in cultured guinea pig tracheal epithelial cells,
studies using anti-ETB immunostaining and [125I]ET-1–
binding assay indicated almost exclusive expression of ETB
by the ciliated columnar cells, and within the cells, localiza-
tion of the receptors in the apical side (6). Cell culture
techniques may affect the expression of ET-1 receptors.
cle cells is associated with a selective increase in the density
and function of the ETBreceptor, a receptor subtype not
present in intact sheep tracheal smooth muscle, with no
change in ETAreceptor density. However, feeding smooth
muscle cells with serum-free medium completely abolishes
the increase in ETBreceptor number (25). Although we
used serum-free media, we cannot rule out that culture
conditions may have affected the expression of endothelin
epithelial cells nor the effect of cell culture conditions on
the distribution of endothelin receptors in human airway
epithelial cells have been determined. Immunolocalization
studies are needed to confirm the apical localization of ETB
receptors in native and cultured human bronchial epithelial
cells, as suggested in this study.
ETAand ETBreceptors may initiate several intracellular
signal transduction events, such as activation of Ca influx,
of phospholipase C, modulation of cAMP, and activation
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 29 2003
of protein kinases (26). In agreement with the present set
of experiments, other investigators found no effect of ET-1
on intracellular Ca concentration in human bronchial epi-
accumulation of intracellular cAMP to the same level as
did IBMX, a phosphodiesterase inhibitor. ET-1 has been
reported not to affect, decrease, or increase intracellular
cAMP, depending on the cell studied. In most expression
systems, ETBis associated with inhibition of adenylate cy-
clase, whereas ETAis associated with stimulation of adenyl-
ate cyclase (26). However, exceptions have been reported:
ETAactivation can lead to inhibition of adenylate cyclase,
tion can lead to activation of adenylate cyclase in specific
cell types, as shown in rabbit tracheal smooth muscle (29,
30). The mechanisms by which ET receptors activate or
inhibit adenylate cyclase activity are also variable among
the tissues studied. Such interactions may occur directly, via
GTP-binding proteins, or through intermediary signaling
components (30, 31). In excised human bronchi, Hay and
colleagues provided evidence for ET-1–induced prostanoid
releasethat wasmediatedvia ETAreceptor activation;how-
ever, their experiments do not indicate whether the source
of prostanoid release was the epithelial cells or other cell
types (32). Takimoto and colleagues found that application
of ET-1 on cultured primary human bronchial epithelial
cells dose-dependently stimulated the secretion of PGE2
via ETA receptor activation (28). Taken together, these
studies indicate that in human bronchial epithelial cells,
ET-1 may provoke the release of prostanoids and that this
release is likely mediated via ETArather than ETBreceptor
activation. Although prostanoids may induce cAMP activa-
tion of Cl secretion in human airways (33), the stimulation
of Cl secretion by ET-1 in our study was probably not
mediated via prostanoids because the effect was (i) medi-
ated via ETBrather than ETAreceptors, and (ii) not inhib-
ited by the cyclooxygenase inhibitor indomethacin.
Sources of production of ET-1 in the lung are multiple.
ET-1 is synthesized and released by endothelial cells, type
act as a paracrine mediator (2, 12). In addition, ET-1 may
also be produced and secreted by airway epithelial cells (2,
12). Animal studies suggest that the nonciliated secretory
cells rather than the ciliated columnar or basal cells are
the major site of ET-1 production in the surface airway
epithelium (6). Interestingly, both animal and human stud-
ies demonstrate that ET-1 can be secreted to both the lumi-
nal and serosal sides of the airway epithelium (2, 6, 34).
These data together with our results imply that ET-1 may
act as an autocrine modulator of airway epithelial Cl trans-
port at the luminal side of the mucosa. Similar autocrine
effects of ET-1 have been implicated in other organs, for
instance in the inhibition of cAMP-dependent anion secre-
tion in human gallbladder epithelial cells (16).
ET-1 is thought to play an important role in the patho-
physiology of various lung diseases such as asthma, chronic
obstructive pulmonary disease, pulmonary hypertension,
and pulmonary fibrosis (2, 12). Production and release of
ET-1 are induced by numerous factors including hypoxia,
bacterial endotoxins, growth factors, and cytokines (2, 12,
34). In asthma, for instance, the production and release of
ET-1 in airways increases dramatically (2, 12). In chronic
obstructive pulmonary disease, sputum levels of ET-1 rise
during exacerbations (35). Our observations, together with
these reports, suggest that enhanced local production of
ET-1 can be expected to participate to the airway hyperse-
cretion observed in patients with inflammatory airway dis-
ease. Hence, antagonists for ETBreceptors and inhibitors
of ET production may provide a useful therapeutic tool for
the treatment of inflammatory airway disease.
In conclusion, ET-1 stimulates transepithelial Cl secre-
tion across cultured human bronchial epithelial cells. This
effect is mediated via ETBreceptors located in the apical
membrane and probably involves activation of the cAMP
pathway. These results support the notion that ET-1 exerts
multiple functions in airways and may be involved in the
pathophysiology of airway inflammatory diseases.
Acknowledgments: This study was supported by grants from the Association
Vaincre la Mucoviscidose, from the Direction a ` la Recherche Clinique, Assis-
tance Publique Ho ˆpitaux de Paris (projet CRC94162), and from CHU Lille
(grant CH&U 99-12352).
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