 |
INTRODUCTION |
In this paper, we investigated the effect of lipoxin
A4
(LXA4)1 on
intracellular [Ca2+] of airway epithelial cell. Lipoxins
are biologically active eicosanoids, originally described to be
produced by leukocytes possessing a 5-lipoxygenase activity, in
the presence of 15-HETE (or 15-HPETE) (1, 2). Lipoxins also result from
the conversion of LTA4 by 12-lipoxygenase activity in
platelets and the 15-lipoxygenase activity in macrophages and
epithelial cells (3-5). The natural isomer lipoxin
A4
(5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid), which
is synthesized at the inflammation site, have been shown to act
as a potent anti-inflammatory mediator in different cellular systems,
affecting leukocyte trafficking by modulating chemotaxis, adhesion,
transmigration, and phagocytic clearance of apoptotic cells (6-9).
LXA4 has been measured in the bronchoalveolar and nasal
lavage fluids of patients with respiratory diseases including asthma (4, 10). Levels of LXA4 are significantly higher in sputum of mild asthmatic patients compared with the levels measured in normal
subjects or asthmatics with a severe form of the disease. Because
LXA4 inhibited interleukin 8 release by peripheral blood mononuclear cells, the reduced levels of LXA4 in severe
asthma might be associated with the persistence of airway inflammation (11). In addition, PMN cells from mild asthmatic patients are able to
generate in vitro larger amounts of LXA4 as
compared with PMN cells obtained from normal individuals (12).
LXA4 causes contraction of pulmonary smooth muscle (13)
although, in asthmatics, LXA4 antagonizes the
broncho-constriction induced by leukotriene C4 (14).
LXA4 stable analogues blocked both airway
hyper-responsiveness and pulmonary inflammation in a murine model of
asthma (15).
The airway epithelial barrier, besides its role in electrolyte and
water balance of the body, also participates in inflammation as a
target cell for inflammatory mediators and by secreting inflammatory cytokines. Intracellular processes such as Ca2+ or
pHi signaling, protein kinase activities, and the airway
neuro-humoral environment finely control the airway epithelial
secretory function altered in respiratory diseases such as asthma and
cystic fibrosis. However, the effect of LXA4 has not been
investigated on the cellular process regulating the human airway
epithelium secretory function, and its potential role in airway
epithelium is not clear. Lipoxins, but particularly LXB4
rather than LXA4, inhibit the proliferation of the human
bronchial epithelial cell line, A549 (16). In addition, the same cell
line did not show appreciable levels of the LXA4 receptor
(17).
Intracellular Ca2+ plays a central role in cellular
functions including inflammatory processes, and interestingly
LXA4 stimulates a [Ca2+]i
increase in rainbow trout leukocytes (18), in human monocytes (19, 20),
and in human neutrophils (21). However, the direct effect of
LXA4 on [Ca2+]i has not been
investigated in epithelial cells. Because
[Ca2+]i is a main regulator of the secretory
functions of the epithelium, the aim of our study was to investigate
the effect of LXA4 on [Ca2+]i and
Cl
secretion through airway epithelium. The present study
is the first report of a rapid and transient
[Ca2+]i increase induced by LXA4
in airway epithelial cells, the description of the cellular mechanisms
generating the response, and its consequence on ionic secretion.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
The human bronchial epithelial
16HBE14o
cell line is derived from surface epithelium of
mainstream, second-generation bronchi (22), forms polarized monolayers
with intact tight junctions, and retains Cl
transport
properties of freshly isolated surface airway epithelial cells (22).
Cells were grown on a collagen/fibronectin coating, in Eagle's minimal
essential medium (BioWhittaker), supplemented with 10% fetal calf
serum, 1% penicillin, 1% streptomycin, and 1%
L-glutamine, and incubated in a 37 °C, 5%
CO2 atmosphere.
For primary culture of human bronchus epithelium, cells were obtained
from surgical biopsies. The bronchial samples were taken from a normal
area of bronchi removed from patients suffering from lung cancer with a
normal lung function (after approval by the ethics committee). After
excision, the bronchial tubes were washed and incubated overnight at
4 °C with 0.38 mg/ml hyaluronidase, 0.75 mg/ml collagenase, 1 mg/ml
protease, and 0.3 mg/ml DNase in RPMI 1640 medium (Invitrogen)
and then filtered through a 70-mm mesh nylon strainer. Pieces of
epithelium retained on the strainer were washed with phosphate-buffered
saline. After centrifugation, epithelial cells were re-suspended in
small airway epithelium basal medium (Clonetics, BioWhittaker, San
Diego, CA) supplemented with 0.5 µg/ml human recombinant epidermal
growth factors, 7.5 mg/ml bovine pituitary extract, 0.5 mg/ml
epinephrine, 10 mg/ml transferin, 5 mg/ml insulin, 0.1 µg/ml retinoic
acid, 6.5 µg/ml triiodothyronine, 50 mg/ml gentamicin, 50 µg/ml
amphotericin B, and 50 mg/ml bovine serum albumin- fatty acid-free.
RNA Extraction and Reverse Transcription-Polymerase Chain
Reaction of LXA4 Receptor--
Total RNA was obtained by
lysing airway epithelial cells using TRIzol reagent (Invitrogen) and
phenol-chloroform extraction and isopropanol precipitation. After DNase
treatment (Invitrogen), 1 µg of total RNA was reversed transcripted
using a Moloney murine leukemia virus (M-MTV RT) reverse
transcriptase (Invitrogen). A first round of PCR was performed using
the sense primer 5'-CAC CAG GTG CTG CTG GCA AG-3' and the anti-sense
primer 5'-AAT ATC CCT GAC CCC ATC CTC A-3' (Applied Biosystems, Foster
City, CA), which were designed to amplify the human peripheral blood
mononuclear cell LXA4 receptor coding region (1,095 bp)
(17). In parallel, the hypoxanthine-guanine
phosphoribosyltransferase gene (213 bp) was used as a
housekeeping gene and amplified using the sense primer 5'-TGT AAT GAC
CAG TCA ACA GGG-3' and antisense primer 5'-TGG CTT ATA TCC AAC ACT
TCG-3' (23). PCRs were carried out with 2 µl of cDNA (28 cycles
of 30 s at 94 °C, 45 s at 64 °C, and 80 s at
72 °C for LXA4 receptor amplification and 35 cycles of
30 s at 94 °C, 50 s at 52 °C, and 80 s at 72 °C
for hypoxanthine-guanine phosphoribosyltransferase amplification) on a
DNA Thermo Cycler (Thermojet; Eurogentec, Angers, France). A nested PCR
was then initiated with 0.5 µl of the first-round PCR products. The
primers used were as follows: 5'-TGC TTG GGG TCA CCT TTG TC-3'
(forward) and 5'-TGA AGC AGA ATT GGC AGC CG-3' (reverse), corresponding to LXA4 receptor primers generating a 928-bp fragment (24). As described by Sodin-Semrl et al. (24), the second set of
LXA4 receptor primers was chosen with minimal homology with
other members of the formyl peptide receptor family and exclude random
crossing with known genomic sequences. Samples were subjected to 28 cycles of 30 s at 94 °C, 45 s at 58 °C, and 80 s
at 72 °C, followed by a final extension step at 72 °C for 5 min.
PCR products were analyzed using electrophoresis in Tris-boric
acid-EDTA buffer with ethidium bromide-stained (0.5 mM) 1%
agarose gel. Bands were captured using an ultrasensitive photon
counting imaging camera (Argus 100; Hamamatsu Photonics, Tokyo, Japan).
Calcium Imaging--
Intracellular [Ca2+] was
determined in 16HBE14o
cells, in primary culture of human
bronchial epithelial cells, or in A549 cells according to a method
previously described (25). Because A549 cells do not form tight
junctions, the calcium experiments were performed on isolated A549
cells. The 16HBE14o
cells and primary culture of human
bronchial epithelial cells grown on permeable support reached
confluency and their highest electrical resistance, on average, 4 days
after plating. Therefore, we arbitrarily distinguished three types of
cell preparations on glass: isolated cells, non-confluent
monolayers (2 days after plating), and confluent monolayers (4 days
after plating). For isolated cells the glass was treated with poly
L-lysine and for monolayers with a fibronectin collagen
solution. The cells were loaded with 5 µM the
Ca2+-sensitive fluorescent probe fura-2 acetoxy-methyl
ester for 30 min, in the dark, at room temperature (22 °C) and were
then washed twice in HEPES-buffered Krebs-Hensleit solution (140 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 280-290 mOsmol). The coverslip covered
with cell monolayer or isolated bronchial cells was mounted on the
stage of an inverted microscope equipped for epi-fluorescence (TE-300,
Nikon, Badhoeve Dorp, Netherlands). Calcium cellular imaging was
performed using the Metafluor Imaging System (Universal Imaging
Corporation). The cell preparation was excited alternatively at
340 and 380 nm using the Optoscan monochromator (Cairn Research Ltd,
Kent, UK). The emission fluorescence produced after fura-2 excitation
was filtered at 510 nm. The transmitted light image was detected using
a Photometrics CoolSNAP-fx video camera (Roper Scientific, Evry,
France) coupled to the microscope. The fluorescence obtained at each
excitation wavelength (F340 and
F380) depends upon the level of Ca2+
binding to fura-2, according to an in vivo calibration
performed using a range of EGTA-buffered Ca2+ solutions of
the fura-2-free acid. The [Ca2+]i was
calculated automatically by a computer program using the Grynkiewicz
equation (26).
Short-circuit Current Measurement--
For transepithelial
transport study, the 16HBE14o
cells were grown on
permeable filters (Costar Snapwell-Clear 3801, 12-mm diameter, 0.4 mm
pore size). Monolayers of 1.2 cm2 exposed surface area were
mounted in a horizontal Ussing chamber between 0 and 3 days after the
electrical resistance reached a maximum value (800-1200 ohms) as
previously described (27). To create a favorable electrochemical
gradient for Cl
secretion, the basolateral side of the
monolayer was bathed in HEPES-buffered Krebs-Hensleit solution, and the
apical was bathed in a low Cl
solution (125 mM sodium gluconate, 15 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 280-290 mOsmol). The spontaneous trans-membrane potential was measured
and clamped to 0 mV by application of a short-circuit current
(Isc) using a voltage clamp model amplifier (EVC 4000, World
Precision Instrument). Frequency of acquisition was every 10 s.
Under these conditions the Isc is a measure of electrogenic
transepithelial ion transfer. The output from the amplifier was
digitized using a PowerLab system (Chart for Windows v4.0, ADInstrument).
Lipoxin--
The LXA4 used in this study was
provided by Calbiochem. The absence of transformation into
all-trans lipoxin isomers was verified using reverse
phase-high pressure liquid chromatography analysis according to
the methods previously described (12). Aliquots of LXA4
solution in ethanol were stored at
80 °C to avoid any damage of
the molecule.
Data Analysis--
The mean [Ca2+]i
variations correspond to the [Ca2+]i
variations between the mean [Ca2+]i measured
during the 2 min prior to exposure to LXA4 and the [Ca2+]i measured at the peak. In each
experiment, the mean [Ca2+]i was obtained
from all cells in the microscope field. Data are shown as the mean ± S.E. of n experiments. Measures of statistical significance were obtained using the Kruskal-Wallis non-parametric test
or the Student's t test for paired data. A
p value <0.05 was deemed to be significant. All statistical
operations were performed using Excel software (Microsoft).
 |
RESULTS |
LXA4 Effect on Intracellular
[Ca2+]--
Exposure of human bronchial epithelial cells
to LXA4 produced a large and transient increase in
[Ca2+]i (Fig.
1, A and B). In
primary culture of human bronchial (PCHB) epithelial non-confluent cell
monolayer (2 days after plating), apical LXA4
(10
8 M) produced an increase in
[Ca2+]i of 526 ± 59 nM
(n = 3, p < 0.001) from a basal value
of 129 ± 35 nM (n = 3). In
16HBE14o
non-confluent cell monolayers or isolated cells
exposure to LXA4 produced a
[Ca2+]i increase of 407 ± 57 nM (n = 6, p < 0.001) from
a basal value of 135 ± 30 nM (n = 6)
and an increase of 510 ± 50 nM (n = 7, p < 0.001) from a basal value of 135 ± 50 nM (n = 7), respectively. In
16HBE14o
monolayers having reached confluency for more
than 4 days, apical exposure to LXA4 did not affect
[Ca2+]i. In A549-isolated cells,
LXA4 did not affect the [Ca2+]i.
However, as a positive control we verified that a further exposure of
A549 cells to apical ATP (10
4 M), known to
stimulate a [Ca2+]i increase, produced the
expected response (Fig. 1C). The dose dependence of the
Ca2+ response to LXA4 in 16HBE14o
cells is shown in Fig. 1D. The larger increase in
[Ca2+]i was obtained using LXA4
at 0.1 µM. One nM of LXA4 did not
produce any detectable change in [Ca2+]i.
Ethanol used as a LXA4 solvent did not affect
[Ca2+]i.

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Fig. 1.
LXA4 effect on
[Ca2+]i. Typical intracellular
[Ca2+]i recording upon exposure to
LXA4 (10 8 M) in non-confluent
PCHB monolayers (A), in non-confluent 16HBE14o
monolayers (B), and in A549-isolated cells (C)
loaded with fura-2 acetoxy-methyl ester (5 µM).
D, effect of increasing doses of LXA4 on
[Ca2+]i variations in non-confluent
16HBE14o cells (D). For each LXA4
concentration, 4 independent experiments have been performed.
LXA4 10 9 M and ethanol used as
the LXA4 solvent did not produce any significant
[Ca2+]i changes. All the other
LXA4 concentrations used produced a significant
[Ca2+]i increase. (**,
p < 0.01; *, p < 0.05, and
NS, non-significant, p > 0.05).
|
|
Calcium Source for LXA4 Response--
To identify the
source of the [Ca2+]i increase induced by
LXA4 we investigated the effect of thapsigargin, a
Ca2+-ATPase pump inhibitor, and removal of external
Ca2+ on 16HBE14o
non-confluent cell
monolayers. As shown in Fig.
2A, thapsigargin (1 µM) produced a slow [Ca2+]i
increase by 165 ± 47 (n = 5) followed by a return
to a plateau value. After 15 min of thapsigargin treatment, the
effect of LXA4 (10
8 M) on
[Ca2+]i was completely abolished. After
thapsigargin, exposure of the 16HBE14o
cells to
LXA4 (10
8 M) did not produce any
significant change in [Ca2+]i
(
[Ca2+]i = 2.5 ± 5 nM,
n = 5, p > 0.1) (Fig. 2, A
and C). Exposure of 16HBE14o
cells to external
Ca2+-free medium did not affect the response to
LXA4. In Ca2+-free solution (0 mM
CaCl2 and 5 mM EGTA), LXA4
(10
8 M) produced a
[Ca2+]i increase by 348 ± 105 nM (n = 5, p < 0.001),
which was not significantly different from the LXA4-induced
Ca2+ increase produced in control conditions
(p > 0.05, Fig. 2, B and C).
Taken together, these results indicate that the response of human
bronchial epithelial cells to LXA4 mainly involvs the Ca2+ release from thapsigargin-sensitive intracellular
stores rather than Ca2+ entry from the extracellular
compartment.

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Fig. 2.
Calcium source of the LXA4
effect. Intracellular [Ca2+]i recording
upon exposure to LXA4 (10 8 M) in
non-confluent 16HBE14o monolayers after thapsigargin (1 µM) treatment (A) or after removal of
Ca2+ (+ 5 mM EGTA) from the external bathing
solution (B). C, statistical representation of
the [Ca2+]i changes upon the two experimental
protocols described above. (**, p < 0.01 and NS,
p > 0.05).
|
|
Effect of (Rp)-cAMP, MDL Hydrochloride, and
Chelerythrine Chloride--
The role of either protein kinase A or
protein kinase C signaling pathways in the mediation of the response
have been investigated using (Rp)-cAMP
(inhibitor of protein kinase A activity), MDL hydrochloride (inhibitor
of adenylate cyclase), and chelerytrine chloride (inhibitor of protein
kinase C activity) on 16HBE14o
non-confluent cell
monolayers. Neither (Rp)-cAMP (200 µM), MDL hydrochloride (50 µM), nor
chelerythrine chloride (10
7 M)
significantly affected the basal level of
[Ca2+]i (
[Ca2+]i = 6 ± 8 nM, n = 4;
[Ca2+]i =
5.6 ± 6.1 nM, n = 3;
[Ca2+]i =
1.4 ± 4 nM,
n = 3, respectively). As shown in Fig.
3, A, B, and
D, (Rp)-cAMP (200 µM)
and MDL (50 µM), largely antagonized the response to
10
8 M of LXA4
(
[Ca2+]i = 26 ± 26 nM,
n = 4;
[Ca2+]i =
142 ± 13 nM, n = 3, respectively).
After chelerythrine chloride (10
7 M), the
Ca2+i response to LXA4
(10
8 M) was not significantly different from
control conditions (
[Ca2+]i = 495 ± 21 nM, n = 3, p > 0.05)
(Fig. 3, C and D).

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Fig. 3.
(Rp)-cAMP, MDL
hydrochloride, and chelerythrine chloride effects. A,
intracellular [Ca2+]i recording upon exposure
to LXA4 in non-confluent 16HBE14o monolayers
during (Rp)-cAMP (200 µM)
treatment. MDL hydrochloride (50 µM) treatment
(B) or chelerythrine chloride (10 7
M) exposure (C). D, statistical
representation of the [Ca2+]i changes upon
the three experimental protocols described above.
|
|
Effects of Pertussis Toxin and Montelukast--
Because A549 cells
do not express detectable levels of LXA4 receptor, the
absence of the effect of LXA4 on
[Ca2+]i in A549 cells suggested that in
16HBE14o
cells the Ca2+ response to
LXA4 is mediated by the LXA4 receptor. Because
the LXA4 receptor have been identified to be coupled to
pertussis toxin-sensitive G-protein, we tested the effect of the toxin
on the Ca2+ response induced by LXA4 in
16HBE14o
-isolated cells. As shown in Fig.
4, A and C, in
16HBE14o
cells pertussis toxin treatment completely
abolished the response to LXA4 at either 10
8
or 10
7 M. LXA4 produced a
non-significant [Ca2+]i variation of
11 ± 15 nM (n = 5, p > 0.1) at 10
8 M and of
2.5 ± 3 nM (n = 5, p > 0.1) at 10
7 M. We also
investigated the eventual role of the cysteinyl leukotriene receptor,
which can be recognized by LXA4 and is also sensitive to
pertussis toxin in the [Ca2+]i response to
LXA4. As shown in Fig. 4, B and C,
montelukast (10
6 M) used as a specific
leukotriene receptor antagonist did not affect the Ca2+
response to LXA4 (10
8 M).

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Fig. 4.
Pertussis toxin and montelukast effects.
Intracellular [Ca2+]i recording upon exposure
to LXA4 of non-confluent 16HBE14o monolayers
treated during 1 h with 2.5 µg/ml of pertussis toxin
(A) or after montelukast (10 6 M)
treatment (B). C, statistical representation of
the [Ca2+]i changes during LXA4
(10 8 M) exposure in control conditions, after
pertussis toxin or montelukast treatment as described above. (**,
p < 0.01 and NS, p > 0.05).
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|
LXA4 Receptor mRNA--
The LXA4
receptor mRNA expression was investigated in three different human
bronchial epithelial cells (the PCHB, the 16HBE14o
, and
the A549 cells) by RT-PCR using specific primers for LXA4 receptor cDNA. Expression of the housekeeping gene
hypoxanthine-guanine phosphoribosyltransferase was determined to
provide an internal control for RT and PCR efficiencies. Peripheral
blood mononuclear cells, from which the LXA4 receptor has
been cloned, were used as positive control (20). Representative
electrophoretic analysis of the PCR products obtained is shown in Fig.
5. LXA4 receptor mRNA
expression was not detected after a first round of RT-PCR amplification
of any of the A549, 16HBE14o
, and PCHB cell mRNAs.
Detection of LXA4 receptor transcripts in human bronchial
epithelial cells required the use of nested PCR, as shown in the
representative electrophoretic analysis in Fig. 5. We
demonstrated that 16HBE14o
and PCHB expressed
LXA4 receptor mRNA, whereas A549 cells did not.

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Fig. 5.
LXA4 receptor mRNA expression
in human bronchial epithelial cells. RT-PCR amplification of a
1095-bp fragment from 16HBE14o , A549 and PCHB
cells was performed using specific primers for LXA4
receptor cDNA. Peripheral blood mononuclear cells were used as
positive control. After 28 cycles of PCR amplification
(top), 0.5 µl of the resulting PCR product were used for a
nested PCR step (28 cycles) using a second set of internal
LXA4 receptor primers amplifying a 928-bp fragment
(middle PCR). PCR products were resolved in 1% agarose
gel.
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|
Effect of LXA4 on Transepithelial Ion
Transport--
Because intracellular [Ca2+]i
is a major regulator of epithelial secretory function we investigated a
possible role for LXA4 on a
Ca2+-dependent Cl
secretion.
LXA4 (10
8 M) applied on the
basolateral side of the monolayer produced a sustained increase by
6 ± 1 µA/cm2, (p < 0.01, n = 6) of the transepithelial short-circuit current (Isc). The Isc increase started between 0 and 5 min after the beginning of LXA4 exposure and reached a
plateau value within 10 min (Fig.
6A). In absence of external
Cl
ions on both side of the epithelium, the response to
LXA4 was completely abolished, indicating that the
LXA4 effect on the Isc was mainly due to
Cl
secretion (Fig. 6B). The role of
intracellular [Ca2+]i on the Isc
increase was investigated using the intracellular calcium chelator, the
BAPTA-AM. As shown in Fig. 6C, BAPTA-AM (10 µM) produced a low decrease (
0.9 ± 0.2 µA/cm2, n = 3) of the initial
Isc, indicating a weak contribution of intracellular
[Ca2+] to the basal trans-epithelial ion transport.
However, after 60 min of treatment with BAPTA-AM, the LXA4
effect on short-circuit current was completely inhibited. These results
indicated that the LXA4-induced short-circuit current
increase is due to the stimulation of a
Ca2+-dependent secretory pathway.

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Fig. 6.
LXA4 effect on short-circuit
current. Basolateral effect of LXA4 on the
transepithelial short-circuit current of 16HBE14o grown
on a permeable filter. The short-circuit current was measured in
control condition (basolateral Krebs-Hensleit solution and apical low
chloride solution) (A) or in Cl -free solution
(B) or in presence of BAPTA-AM (10 µM) on both
sides of the monolayer.
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|
 |
DISCUSSION |
The present study is the first description of the rapid
[Ca2+]i increase induced by LXA4
in human airway epithelial cells including the description of the
cellular mechanism involved in the response. The stimulation of
a [Ca2+]i increase by LXA4 has
been reported in rainbow trout leukocytes (18), in human monocytes (19,
20), and in human neutrophils (21). In airway epithelial cell,
identification of the intracellular calcium stores as the main source
of the LXA4-induced [Ca2+]i
increase is consistent with the previous report of an intracellular calcium mobilization induced by LXA4 in other cell types
(19, 20). In addition, the murine LXA4 receptor recently
identified and cloned was also shown to induce the production of
inositol triphosphate, a cellular event related to
Ca2+ mobilization (28). There is no previous report
of a [Ca2+]i increase induced by
LXA4 in epithelial cells. In T84 cells, an intestinal
epithelial cell line, LXA4 inhibited the transmigration of
PMN by decreasing the Ca2+ response to the
pathogen-elicited epithelial chemo-attractant, but the direct effect of
LXA4 on [Ca2+]i was not reported
(29).
The [Ca2+]i response to LXA4 was
dose-dependent with a maximal response measured at
10
7 M, which is consistent with the
[Ca2+]i response to LXA4 in other
cell types. A maximal effect was obtained at 10
6
M in human neutrophils and human monocytes and at
10
8 M in rainbow leukocytes (18-21). In
human monocytes the [Ca2+]i response was
mediated by stimulation of the LXA4 receptor (20).
The LXA4 receptor has been cloned in human leukocytes (30)
and in intestinal epithelial cell lines (17). We found mRNA expression of the LXA4 receptor in the
16HBE14o
cell line and in human bronchial epithelial
cells in culture, but not in A549 cells. The detection of the
LXA4 receptor mRNA in human airway epithelial cells is
consistent with the detection of LXA4 receptor cDNA in
murine lung (28). Our results are in accordance with the previous
publication (31) reporting that the human bronchial epithelial cell
line A549 did not show appreciable levels of LXA4 receptor
cDNA, which we interpreted as a cell line characteristic rather
than a tissue specificity of the LXA4 receptor expression. Because there is not detectable LXA4
receptor expression in A549 cells, we considered this cell line as a
negative control. Therefore, the relation between the LXA4
receptor mRNA detection and the [Ca2+]i
response strongly suggests that the LXA4 receptor was
involved in the Ca2+ response. Moreover, because the
LXA4 receptor activation has been shown to be associated to
pertusssis toxin-sensitive G-protein, the sensitivity of the
LXA4-induced [Ca2+]i response to
pertussis toxin is also consistent with a role for LXA4
receptor in the response. It has been reported that in rat glomerular
mesengial cells, LXA4 binds the LTD4 receptor, which is also coupled to a G-protein (32). However, we have shown that
the LTD4 antagonist montelukast did not affect the [Ca2+]i response to LXA4 in
airway epithelium, which indicates that the
[Ca2+]i response to LXA4 is not
mediated by the LTD4 receptor (34). This last result
constitutes further evidence for the involvement of the
LXA4 receptor in the Ca2+ response to
LXA4 in airway epithelial cell.
Because LXA4 is a lipophylic molecule, the side of
epithelium exposure to LXA4 should not affect the response.
However, the absence of Ca2+ response to apical exposure to
LXA4 of highly confluent 165HBE14o
monolayers
compared with the response obtained in isolated cells or non-confluent
cell monolayers could be explained by the lack of accessibility of the
extracellular side of the receptor. This would suggest a basolateral
location of the LXA4 receptor. In addition this observation
is consistent with the recent report (35) of a preferential lateral
location of the LXA4 receptor in intestinal epithelial
cells. Therefore, we tested the modulation of the Isc by
LXA4 applied on the basolateral side.
Because the role of LXA4 in protein kinase C activation
have been largely reported, we investigated the involvement of protein kinase C activity in the generation of the Ca2+ response
(36-39). The ineffectiveness of chelerythrine chloride, a protein
kinase C inhibitor, in the Ca2+ response to
LXA4 suggests that protein kinase C activity is not involved in the generation of the [Ca2+]i
increase. However, the stimulation of protein kinase C activity by
LXA4 might involve an initial Ca2+ increase, as
described in the present study. The role of protein kinase A activity
has also been investigated. The inhibitory effect of
(Rp)-cAMP and MDL hydrochloride suggested that,
in airway epithelium, the Ca2+ response to LXA4
involved stimulation of adenylate cyclase and protein kinase A
activities. In contrast, the LXA4 effect on actin rearrangement in monocytes and macrophages is mimicked by the protein
kinase A inhibitor and inhibited by the cell permeant cAMP analogue
8-Br-cAMP (40).
Intracellular [Ca2+] plays a central role in epithelial
cellular function such as ion or protein secretion. The
LXA4-induced [Ca2+]i response
might be involved in eicosanoid production by epithelium, which is
known to depend on [Ca2+]i (41-43). More
specifically, a [Ca2+]i increase is necessary
for lipoxygenase activity in the production LXA4 from its
substrate (12, 42, 43). Therefore, the LXA4-induced [Ca2+]i increase might play a central role in
an autocrine production of LXA4 and constitute an
amplificatory mechanism for the biosynthesis of LXA4 itself.
In addition, our results suggested that the Cl
secretion
induced by LXA4 is one of the functional consequences of
the LXA4-induced [Ca2+]i
increase. This would indicate that, in airway epithelial cells, beside
its anti-inflammatory role LXA4 is involved in ionic
transport regulation. This might have important clinical implications,
in particular in the treatment of respiratory diseases associated with
ion transport dysfunction, and necessitating anti-inflammatory treatment such as cystic fibrosis and asthma.