Intracellular calcium oscillations induced by ATP in airway
epithelial cells
John H.
Evans and
Michael J.
Sanderson
Department of Physiology, University of Massachusetts Medical
School, Worcester, Massachusetts 01655
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ABSTRACT |
In airway epithelial cells, extracellular ATP
(ATPo) stimulates an initial
transient increase in intracellular
Ca2+ concentration that is
followed by periodic increases in intracellular Ca2+ concentration
(Ca2+ oscillations). The
characteristics and mechanism of these ATP-induced Ca2+ responses were studied in
primary cultures of rabbit tracheal cells with digital video
fluorescence microscopy and the
Ca2+-indicator dye fura 2. The
continual presence of ATPo at
concentrations of 0.1-100 µM stimulated
Ca2+ oscillations that persisted
for 20 min. The frequency of the Ca2+ oscillations was found to be
dependent on both ATPo
concentration and intrinsic sensitivity of each cell to
ATPo. Cells exhibited similar
Ca2+ oscillations to extracellular
UTP (UTPo), but the oscillations typically occurred at lower UTPo
concentrations. The ATP-induced Ca2+ oscillations were abolished
by the phospholipase C inhibitor U-73122 and by the endoplasmic
reticulum Ca2+-pump inhibitor
thapsigargin but were maintained in
Ca2+-free medium. These results
are consistent with the hypothesis that in airway epithelial cells
ATPo and
UTPo act via P2U purinoceptors to
stimulate Ca2+ oscillations by the
continuous production of inositol 1,4,5-trisphosphate and the
oscillatory release of Ca2+ from
internal stores. ATP-induced Ca2+
oscillations of adjacent individual cells occurred independently of
each other. By contrast, a mechanically induced intercellular Ca2+ wave propagated through a
field of Ca2+-oscillating cells.
Thus Ca2+ oscillations and
propagating Ca2+ waves are two
fundamental modes of Ca2+
signaling that exist and operate simultaneously in airway epithelial cells.
inositol 1,4,5-trisphosphate; calcium waves; uridine
5'-triphosphate; adenosine 5'-triphosphate; cell
signaling
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INTRODUCTION |
OSCILLATORY CHANGES in intracellular
Ca2+ concentration
([Ca2+]i),
or Ca2+ oscillations, occur in a
variety of nonexcitable cell types (47). Commonly,
Ca2+ oscillations arise after the
activation of phospholipase C (PLC) by agonists binding to cell surface
receptors (1). This leads to the production of inositol
1,4,5-trisphosphate (IP3)
followed by Ca2+ mobilization from
internal stores via the IP3
receptor (IP3R), itself a
Ca2+ channel, and to an increase
in
[Ca2+]i.
Ca2+ feedback inhibition of the
IP3R results in cessation of
Ca2+ release (3), and this
together with the sequestration and extrusion of
Ca2+ from the cytoplasm by pumps
into the endoplasmic reticulum or across the plasma membranes,
respectively, leads to a decline in
[Ca2+]i.
Once the
[Ca2+]i
falls to a permissive level, repetitive cycles of
[Ca2+]i
increase and decline are maintained by an elevated intracellular IP3 concentration
([IP3]i)
(13, 15). Thus Ca2+ oscillations
are dependent on the action of both
[Ca2+]i
and
[IP3]i
on the IP3R. Experimental data
show that the frequency of Ca2+
oscillations are dependent on
[IP3]i
and, indirectly, agonist concentration (15). Mathematical modeling of
Ca2+ oscillations as a function of
[IP3]i
and
[Ca2+]i
agrees with the experimental findings (43).
Extracellular ATP (ATPo) serves
as an agonist in many organs and tissues (12) and induces an initial
transient increase in
[Ca2+]i
followed by Ca2+ oscillations in a
number of cell types including astrocytes (49), smooth muscle cells
(24), granulosa luteal cells (44), Madin-Darby canine kidney (MDCK)
cells (35), chondrocytes (8), bile duct epithelial cells (29), and
megakaryocytes (46). In airway cells,
ATPo mobilizes
[Ca2+]i
via PLC by activating P2U receptors (12). P2U receptors
are expressed at the apical pole of rat airway epithelial cells and respond to extracellular UTP
(UTPo) as well as to
ATPo (19, 26).
ATPo and
UTPo may serve as important
physiological factors in the airway lumen because airway cells are
reported to release ATP and UTP in response to stretch (14) and via the
cystic fibrosis transmembrane conductance regulator (9). Released
ATPo or
UTPo may act in an autocrine or
paracrine fashion to mediate
Cl
secretion (42) and,
possibly, ciliary beat frequency (16, 50).
The spatial organization of
[Ca2+]i
responses to an agonist are also important in coordinating
Ca2+-mediated activity at the
level of tissues or organs. For example, synchronous
Ca2+ oscillations have been
reported in tissues such as pancreatic acini (45), islets of Langerhans
(2), lung capillary endothelium (51), and liver lobules (30, 33) and in
cultures of hepatocytes (48), MDCK cells (35), and chondrocytes (8).
The synchrony of Ca2+ oscillations
in the above systems invariably relies on gap junction communication
between contacting cells, and the synchrony is lost when communication
is disrupted. In perfused pancreatic acini (45) and other confluent
cell cultures, such as arterial smooth muscle cells (24) and MDCK cells
(35), however, no synchrony of
Ca2+ oscillations between
contacting cells is observed. Thus between cell types and experimental
conditions, the spatial coordination of
Ca2+ oscillations differs. These
differences may then be exploited to investigate different mechanisms
of intercellular communication. Because both
Ca2+ and
IP3 can act as intercellular
messengers (36), it remains unclear whether
Ca2+ or
IP3 is the messenger communicated
though the gap junctions to initiate intercellular
Ca2+ waves or whether the
intercellular messenger employed is cell-type or condition specific. In
addition to gap junction communication, cell messengers can be
communicated extracellularly (20, 41). In primary cultures of airway
epithelial cells, the intercellular diffusion of
IP3 is consistent with the
observed propagation of intercellular
Ca2+ waves under a number of
experimental conditions (5).
Although Ca2+-sensitive cell
functions are often mediated by oscillatory rather than prolonged
sustained increases in
[Ca2+]i
(32), only a few brief reports have shown
Ca2+ oscillations in ciliated
airway epithelial cells (21, 31, 37), goblet cells (31), or airway
gland cells (25). Using digital microscopy techniques and the
Ca2+ indicator fura 2, we
investigated the temporal aspects of the [Ca2+]i
response to ATPo and
UTPo in airway epithelial cells
and describe here the mechanism by which
Ca2+ oscillations are initiated
and maintained. We found that ATP-induced Ca2+ oscillations in airway
epithelial cells were dependent on the ATPo concentration
([ATP]o), were
mediated by G protein-coupled receptors involving an
IP3 signaling pathway, and were
not communicated to adjacent cells. Thus airway epithelial cells
exhibit two fundamental modes of
Ca2+ signaling, intracellular
Ca2+ oscillations and
intercellular Ca2+ waves, which
may occur simultaneously within a cell.
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MATERIALS AND METHODS |
The techniques for culturing airway epithelial cells, mechanically
stimulating individual cells, and the measurement of
[Ca2+]i
by fluorescent video microscopy have been described in detail elsewhere
(7, 23, 38-40) and will be only briefly reviewed here.
Cell culture. Primary cultures of
airway epithelial cells were prepared from the tracheae of New Zealand
White rabbits as previously described (11) except that the collagen on
the coverslips was fixed with formaldehyde instead of glutaraldehyde.
After dissection of the epithelial mucosa from the trachea, the tissue
was cut into ~0.5-mm squares, placed onto collagen-coated coverslips, and cultured in DMEM supplemented with 10% fetal bovine serum, 10 mM
HEPES, and antibiotics-antimycotics at 37°C in 10%
CO2 for 5-9 days.
Measurement of
[Ca2+]i.
Cells were loaded with fura 2 by incubation at 37°C for 1 h in 5 µM fura 2-AM (Calbiochem) in
Ca2+ (1.3 mM)-containing Hanks'
buffered salt solution without phenol red (HBSS; GIBCO BRL) and
additionally buffered with 25 mM HEPES (HHBSS, pH 7.4).
The cells were washed and allowed to incubate at room temperature for
30 min to allow for complete deesterification of the fura 2-AM. In
Ca2+-free experiments,
Ca2+/Mg2+-free
Dulbecco's phosphate-buffered saline (DPBS; GIBCO BRL) was used in
place of HHBSS. Cells were visualized with a Nikon inverted microscope
equipped with fluorescence optics and a ×40 objective lens.
Fluorescence was detected with a silicon-intensified target camera,
recorded with an optical memory disk recorder, and digitized by
computer (7, 38). Images of
[Ca2+]i
were calculated by single-wavelength recordings referenced to
ratiometric measurements (23). Initial
[Ca2+]i
reference images were based on 10 frames recorded at 340 and 380 nm.
Changes in
[Ca2+]i
were recorded by monitoring changes in fluorescence with an illumination wavelength of 380 nm. Additional reference images were
taken at 340 nm every 30 s.
[Ca2+]i
was calculated from the change in fluorescence intensity at 380 nm (7,
23). All images were subjected to background subtraction and correction
for shading and bleaching. For plots of
[Ca2+]i
versus time, single points encompassing an area of 2.1 × 2.3 µm
were selected from the cells of interest, and
[Ca2+]i
was calculated only at those points. Time-lapse recordings were made at
2 images/s (each frame recorded at 30 frames/s).
Drug application. ATP and UTP (Sigma)
were dissolved in distilled H2O at
10 mM and stored in aliquots at
20°C. Desired final concentrations were made by dilution of the stock in HHBSS or Ca2+/Mg2+-free
DPBS. Thapsigargin, U-73122, and U-73343 (BIOMOL) were dissolved in
dimethyl sulfoxide (DMSO; 1, 5, and 7.5 mM, respectively), divided into
aliquots, and stored at
20°C. Final concentrations were made by
dilution in HHBSS. Controls for thapsigargin experiments were performed
in 0.1% DMSO. Two hundred microliters of the required experimental
solution were exchanged for the 200 µl of HHBSS in the cell chamber
for each experiment. Between trials, the cells were washed with >3 ml
(>15 volumes) of HHBSS. In all experiments, the cells were allowed to
recover for >15 min between trials or between control and
experimental conditions.
Mechanical stimulation. Mechanical
stimulation of a single cell was performed by brief displacement of the
apical surface of the cell membrane with a glass micropipette
(~1-µm tip diameter) for 100 ms. The magnitude and duration of the
membrane displacement was controlled by applying a voltage pulse with a
Grass stimulator to a piezoelectric crystal to which the micropipette
was attached (40).
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RESULTS |
Response to ATPo.
The Ca2+ response of airway
epithelial cells to ATPo was
quantified with digital video microscopy in confluent primary cell cultures (5-9 days old) loaded with the
Ca2+ indicator fura 2. The
application of ATPo (0.1-100
µM) induced an initial Ca2+
transient in >90% of cells in the field of view. However, the magnitude of this
[Ca2+]i
increase varied greatly from cell to cell, but in general, it increased
with increasing
[ATP]o. After the
initial
[Ca2+]i
increase induced by ATPo,
7-36% of all the cells, depending on the
[ATP]o, displayed
periodic Ca2+ oscillations (Fig.
1). These
Ca2+ oscillations ceased
immediately on ATPo washout (data
not shown). The greatest number of cells displayed
Ca2+ oscillations in response to 5 µM ATP (n = 386-503
cells, 3-4 experiments; Fig.
2A).
These Ca2+ oscillations were
initiated 15 s to several minutes after the initial
[Ca2+]i
transient and consisted of a sharp increase in
[Ca2+]i
followed by a slower
[Ca2+]i
decline; however, the
[Ca2+]i
increases of the Ca2+ oscillations
were smaller than that of the initial
Ca2+ transient. In many
experiments, the initial two to three
Ca2+ oscillations were
characterized by a higher frequency and were initiated from a higher
baseline
[Ca2+]i
than subsequent oscillations.

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Fig. 1.
Response of individual airway epithelial cells to extracellular ATP
(ATPo). Representative traces of
intracellular Ca2+ concentration
([Ca2+]i)
changes with respect to time of 3 different cells exposed to increasing
ATPo concentration
([ATP]o; 0.1-100
µM) are shown. Sensitivities of cells were determined by minimum
[ATP]o value required
to elicit multiple Ca2+
oscillations. A: high ATP-sensitive
cells displayed multiple Ca2+
oscillations at ~0.5 µM ATP. B:
intermediate ATP-sensitive cells displayed
Ca2+ oscillations at ~5 µM
ATP. C: low ATP-sensitive cells
displayed Ca2+ oscillations at
~50 µM ATP. Characteristics of
Ca2+ oscillation responses to
ATPo were similar between each ATP
sensitivity category; frequency of
Ca2+ oscillations increased with
increased [ATP]o
until, at high relative
[ATP]o values,
Ca2+ oscillations ceased and
[Ca2+]i
remained elevated. Traces are representative of data sets of 265 cells
analyzed from 5 independent experiments.
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Fig. 2.
Summary of Ca2+ response of airway
epithelial cells to ATPo.
A: airway epithelial cells displayed
greatest Ca2+ oscillatory behavior
at 5 µM ATP. [ATP], ATP concentration.
B: in a single culture, average number
of Ca2+ oscillations/cell (defined
as average number of
[Ca2+]i
increases/cell during a 5-min recording period after initial
[Ca2+]i
increase) increased with increasing
[ATP]o values.
However, [ATP]o value
generating the greatest number of
Ca2+ oscillations/cell was
dependent on ATPo sensitivity of
cell. C: no. of cells in each ATP
sensitivity category in a single cell culture (same culture as in
B). Intermed, intermediate. Data in
A are from 388-503 cells from
3-5 experiments analyzed at each
[ATP]o value. Data in
B and
C are from a single experiment with 63 cells analyzed.
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Frequently, each Ca2+ oscillation
occurred as an intracellular Ca2+
wave that spread across the individual cell. The region from which the
Ca2+ wave initiated and the
direction of the wave propagation was constant for each intracellular
Ca2+ oscillation and was unique to
each individual cell. The relationship of the
Ca2+ wave initiation site to a
specific cell organelle or structure was not determined but may result
from the heterogeneity in the distribution and sensitivity of the
Ca2+ pools or
IP3Rs. This relationship will be
the subject of a future study. Simultaneous phase-contrast imaging also
demonstrated that the ciliary beat frequency of the cells increased and
decreased in concert with the
[Ca2+]i
changes of each Ca2+ oscillation,
and studies correlating the
[Ca2+]i
and ciliary beat frequency changes also need to be completed.
Dose-response experiments revealed that individual cells within a
population exhibited differing sensitivities to
ATPo and that this determined the
characteristics of the
Ca2+-oscillatory behavior of each
cell. Cells could be categorized as having a high, intermediate, or low
sensitivity to ATPo. Within each
category, four basic responses to
ATPo were well identified (Fig.
1). These consisted of 1) a single
Ca2+ transient after exposure to
ATPo;
2) the initiation of a few irregular
Ca2+ oscillations;
3) the initiation of well-defined,
multiple Ca2+ oscillations; and
4) a prolonged elevation of the
baseline
[Ca2+]i
after the initial spike. For each cell, this range of responses required a 5- to 10-fold increase in
ATPo. For example, a cell with
high sensitivity to ATPo (Fig.
1A) responded to 0.5 or 1 µM ATP
with regular, periodic Ca2+
oscillations. However, the same cell responded to 5 µM ATP with an
initial
[Ca2+]i
transient followed by a prolonged elevated
[Ca2+]i
without any subsequent Ca2+
oscillations. By contrast, a cell with an intermediate sensitivity to
ATPo (Fig.
1B) responded to 5 µM ATP with
well-defined Ca2+ oscillations and
required 50 µM ATP to induce an elevated baseline of
[Ca2+]i
without Ca2+ oscillations. On the
other hand, a cell with low sensitivity to
ATPo required 50 µM ATP to
induce regular Ca2+ oscillations
(Fig. 1C).
These differing Ca2+-oscillatory
responses to ATPo are summarized
in Fig. 2. The greatest number of cells (46% of the culture) was
classified as having an intermediate sensitivity to
ATPo, and these cells displayed
the greatest number of Ca2+
oscillations in response to 10 µM ATP. Cells classified as having either a high (32% of the culture) or low sensitivity (22% of the
culture) to ATPo displayed the
greatest number of Ca2+
oscillations in response to 1 or 100 µM ATP, respectively. Therefore, in a single culture, individual cells displayed similar
[Ca2+]i
responses to ATPo stimulation but
over a 100-fold concentration range.
Response to UTPo.
In an earlier study by Hansen et al. (16), the
[Ca2+]i
response of tracheal epithelial cells to
ATPo was blocked by the antagonist suramin, suggesting P2 purinergic-receptor involvement. Because P2U
receptors exhibit a greater or equal selectivity for
UTPo over
ATPo (12), we determined the
[Ca2+]i
response of cultured epithelial cells to
UTPo. The
[Ca2+]i
response of individual cells to 0.1 µM
UTPo or
ATPo is shown in Fig.
3. In one cell (Fig.
3A),
UTPo elicited an initial
[Ca2+]i
transient, whereas ATPo had no
effect on the
[Ca2+]i.
In another cell (Fig. 3B), the
initial
[Ca2+]i
transient in response to UTPo was
followed by a single Ca2+
oscillation, whereas ATPo elicited
only an initial
[Ca2+]i
increase. However, the initial
[Ca2+]i
transients were virtually identical for both agonists. In a third cell
(Fig. 3C),
UTPo elicited a higher frequency
of Ca2+ oscillations compared with
that elicited by ATPo.

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Fig. 3.
Differential response of individual cells to extracellular UTP
(UTPo) and
ATPo. Cultured cells were
sequentially exposed to 0.1 µM ATP and 0.1 µM UTP or vice versa.
Traces of
[Ca2+]i
change over time are representative of individual cells in which
UTPo but not
ATPo elicited an initial
[Ca2+]i
increase (A) or initiated a
Ca2+ oscillation subsequent to the
initial
[Ca2+]i
increase (B).
C: trace is representative of cells in
which UTPo initiated a higher
frequency of Ca2+ oscillations
compared with that initiated by
ATPo. Traces are representative of
229 cells analyzed from 4 experiments.
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Of cells displaying an
[Ca2+]i
increase in response to 0.1 µM UTP, 91% of these cells exhibited an
[Ca2+]i
increase in response to 0.1 µM ATP
(n = 229 cells, 4 experiments; Fig.
4A).
Less than 2% of cells exhibited an
[Ca2+]i
increase in response to ATPo but
not to UTPo. Also, 0.1 µM UTP
initiated Ca2+ oscillations in
59% of cells compared with 40% with 0.1 µM ATP (Fig.
4A), and the cells stimulated with
UTPo showed a greater number of
oscillations than those stimulated with
ATPo (Fig.
4B). These responses to
ATPo or
UTPo were independent of the order of agonist challenge.

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Fig. 4.
Summary of differential responses to
ATPo and
UTPo.
A: of the cells which exhibited at
least 1 [Ca2+]i
increase in response to 0.1 µM UTP, 91% also responded to 0.1 µM
ATP. Only 4 of 229 cells from 4 experiments that responded to
ATPo failed to respond to
UTPo. Of the responding cells,
59% of cells exhibited oscillations in
UTPo, whereas only 40% exhibited
oscillations in ATPo.
B: average no. of oscillations/cell
induced by UTPo was higher than
that induced by ATPo in the 5-min
recording period.
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Ca2+
oscillations and PLC activity.
The stimulation of
[Ca2+]i
increase by ATPo and
UTPo via P2U receptors is believed
to involve the release of Ca2+
from IP3-sensitive
Ca2+ stores after the activation
of PLC and the production of IP3 (12). In support of this hypothesis, Hansen et al. (16) found that
neither ryanodine nor caffeine increases
[Ca2+]i
in airway epithelial cells and suggested that the internal Ca2+ release does not appear to
involve a ryanodine receptor-mediated Ca2+-induced
Ca2+ release. The involvement of
PLC in the response of epithelial cells to
ATPo is supported by the finding
that an increase in [Ca2+]i
was abolished by the aminosteroid U-73122, a PLC inhibitor, whereas its
inactive analog U-73343 had no effect (17). To determine whether PLC
activation was part of the mechanism of ATP-induced Ca2+ oscillations, we investigated
the effect of U-73122 on ATP-induced Ca2+ oscillations.
Incubation of cells for 20 min in 10 µM U-73122 before application of
5 µM ATP completely abolished the initial
[Ca2+]i
transient as well as the subsequent
Ca2+ oscillations in the cells
(data not shown). A 20-min incubation in 10 µM U-73343 had no effect
on the initial
[Ca2+]i
response or subsequent Ca2+
oscillations (data not shown). Because it is possible that
Ca2+ oscillations require an
initial PLC-dependent
[Ca2+]i
increase but do not require continual activity of PLC, we added 10 µM
U-73122 to cells exhibiting ongoing ATP-induced
Ca2+ oscillations. ATP-induced
Ca2+ oscillations quickly ceased
on addition of U-73122 (Fig.
5A). The
addition of U-73343 had no effect on ongoing
Ca2+ oscillations
(n = 83 cells, 3 experiments; Fig.
5B).

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Fig. 5.
Effect of phospholipase C inhibitor U-73122 and an inactive analog,
U-73343, on
[Ca2+]i
changes induced by ATPo.
A: U-73122 was added to cells 2 min
after exposure to 5 µM ATP. Trace shows that
ATPo induced a characteristic
Ca2+ oscillation pattern and that
Ca2+ oscillations ceased after
addition of U-73122. B: similar
experiment shows that U-73343 had no effect on
Ca2+ oscillations
(n = 83 cells from 3 experiments).
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Ca2+
oscillations and the release of internal
Ca2+ stores.
IP3-mediated
Ca2+ oscillations typically are
dependent on Ca2+ release from
internal stores and are independent of the extracellular Ca2+ concentration
([Ca2+]o).
To investigate the dependence of ATP-induced
Ca2+ oscillations on
[Ca2+]o,
cells were stimulated with 5 µM ATP in
Ca2+/Mg2+-free
DPBS. Because extracellular unbound EGTA has been shown to interfere
with Ca2+ release from
intracellular stores in airway epithelial cells (18) and because the
Ca2+ response to histamine in
airway cells was essentially the same in nominally
Ca2+-free medium, medium
containing 1 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and medium with an
[Ca2+]o
of ~7 nM (18), we decided to use a nominally
Ca2+-free medium for these
experiments. Ca2+ oscillations
induced by ATPo occurred in the
absence of extracellular Ca2+
(n = 33 cells, 3 experiments; Fig.
6A).
Treatment with
Ca2+/Mg2+-free
DPBS alone failed to induce Ca2+
oscillations (Fig. 6B).

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Fig. 6.
Effect of low extracellular Ca2+
concentration on
[Ca2+]i
changes induced by ATPo.
A: cells were washed once in
Ca2+/Mg2+-free
Dulbecco's PBS (DPBS) and then stimulated with 5 µM ATP in
Ca2+/Mg2+-free
DPBS. Trace shows that ATPo
induced a characteristic Ca2+
oscillation pattern in absence of extracellular
Ca2+.
B: similar experiment shows that in
the same cell
Ca2+/Mg2+-free
DPBS alone failed to induce Ca2+
oscillations (n = 33 cells from 3 experiments).
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To investigate further the dependence of
Ca2+ oscillations on internal
Ca2+ stores, we used the
Ca2+-ATPase inhibitor
thapsigargin. Prolonged incubation (20 min) of cells with 1 µM
thapsigargin elevated the baseline
[Ca2+]i
in the cells and abolished the
Ca2+ response of the cells to 5 µM ATP (data not shown). Again,
Ca2+ oscillations may be dependent
on an initial Ca2+ increase but
independent of internal Ca2+
pools. To investigate this possibility, cells were exposed for a short
time (1 min) to thapsigargin before stimulation with
ATPo. This short-term exposure to
thapsigargin stimulated a small rise in
[Ca2+]i
but allowed a large ATP-induced
[Ca2+]i
increase (n = 91 cells, 4 experiments;
Fig.
7A).
Baseline
[Ca2+]i
levels remained elevated in these cells, and the cells failed to
exhibit Ca2+ oscillations. An
identical treatment of cells with DMSO, the vehicle for thapsigargin,
resulted in a normal Ca2+ response
to ATPo treatment (Fig.
7B). To determine whether internal release of Ca2+ is the sole
mechanism driving ATP-induced Ca2+
oscillations, we treated cells displaying ATP-induced
Ca2+ oscillations with
thapsigargin. After the addition of thapsigargin to
Ca2+-oscillating cells, the
amplitude of the Ca2+ oscillations
decreased and the baseline
[Ca2+]i
increased with each successive oscillation until oscillations ceased
(n = 154 cells, 5 experiments; Fig. 7,
C and
D).

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Fig. 7.
Effect of thapsigargin on
[Ca2+]i
changes induced by ATPo.
A: cells were exposed to 5 µM ATP
after a 60-s incubation with thapsigargin. Trace shows that after a
transient increase,
[Ca2+]i
remained elevated. C: cells exposed to
5 µM ATP were treated with thapsigargin at 60 s. Trace shows that
thapsigargin induced a diminution of
Ca2+ oscillation amplitude, with a
concomitant elevation in baseline
[Ca2+]i
followed by a cessation of oscillations at an elevated baseline
[Ca2+]i.
In similar experiments where 0.1% DMSO was added 60 s before
(B) or after
(D) exposure to 5 µM ATP,
characteristic ATP-induced
Ca2+ oscillations were observed.
Traces in A and
B are representative of 108 cells
analyzed from 3 independent experiments and in
C and
D are representative of 154 cells
analyzed from 5 independent experiments.
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Spatial characteristics of
Ca2+
oscillations.
To ascertain the ability of cells exhibiting
Ca2+ oscillations to influence the
Ca2+ activity of neighboring
cells, we analyzed the temporal and spatial [Ca2+]i
responses of cells to ATPo in
confluent cultures. Pseudocolor images of the
[Ca2+]i
change in airway epithelial cells exposed to 5 µM ATP (Fig. 8) showed that after an initial
[Ca2+]i
increase stimulated by the addition of
ATPo (Fig.
8A), many cells displayed repetitive
increases in
[Ca2+]i
of differing frequency and amplitude (Fig. 8,
B-I).
The asynchronous nature of the
Ca2+ oscillations is exemplified
by five adjacent cells (Fig. 8,
) that exhibited
Ca2+ oscillations independently
from one another.

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Fig. 8.
Pseudocolor images of
[Ca2+]i
change in cells exposed to ATPo.
Airway epithelial cells in a confluent culture were treated with 5 µM
ATP and responded with an initial increase in
[Ca2+]i
(A). After recovery from initial
[Ca2+]i
increase (B and
C), several cells initiated
Ca2+ oscillations with frequencies
and amplitudes that were intrinsic to each cell
(D-I).
Individual nature of Ca2+
oscillations is highlighted by asynchrony of
Ca2+ oscillations in 5 adjacent
cells ( ). White lines, approximate cell outlines. Times are relative
to ATPo addition.
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Graphic representations of the
[Ca2+]i
changes in four adjacent cells (Fig. 9)
show that all cells responded to the addition of 5 µM ATP with a
nearly simultaneous
[Ca2+]i
increase. However, the patterns of
Ca2+ oscillations differed greatly
between cells. For example, the first
Ca2+ oscillation in
cell B (central cell) occurred at
~40 s (Fig. 9, line 1) and failed
to propagate to cell C. This
Ca2+ oscillation preceded the
first Ca2+ oscillation of
cell A by ~50 s and followed the
first Ca2+ oscillation of
cell D by ~8 s. Similarly, the
second Ca2+ oscillation in
cell B (Fig. 9, line
2) preceded the first
Ca2+ oscillation in
cell A by ~6 s and followed the
second Ca2+ oscillation in
cell D by ~20 s. By 150 s,
cells
A-D have
experienced 1, 3, 0, and 4 Ca2+
oscillations, respectively.

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Fig. 9.
Temporal and spatial organization of ATP-induced
Ca2+ oscillations. Outlines
(top) and
Ca2+ traces
(bottom) of 4 cells treated with 5 µM ATP are shown. In response to
ATPo (line
A), cell B exhibited
characteristic Ca2+ oscillations
and was bordered by cells that exhibited
Ca2+ oscillations of lower
(cell A) and higher
(cell D) frequencies and by a cell
that exhibited no oscillations (cell
C). Ca2+
oscillations in cell B
(lines
1-3) were
temporally distinct from Ca2+
oscillations in neighboring cells.
|
|
Intercellular
Ca2+ signaling.
In a previous study (6), it has been demonstrated that in
response to mechanical stimulation, airway epithelial cells propagated increases in
[Ca2+]i
similar to those associated with
Ca2+ oscillations to adjacent
cells as intercellular Ca2+ waves.
The mechanism responsible for the intercellular waves was proposed to
be the diffusive spread of IP3
rather than of Ca2+ between
adjacent cells via gap junctions. Consequently, the asynchronous nature
and failure of ATP-induced Ca2+
oscillations to influence the Ca2+
activity of adjacent cells may result either from an inherent but
independent regulation of cell function or, alternatively, from a lack
of gap junction communication between cells. To explore this hypothesis
and to estimate the extent of cell-cell communication, the ability of
cells displaying ATP-induced Ca2+
oscillations to propagate intercellular
Ca2+ waves was tested.
Figure 10 shows pseudocolor
images of the change in
[Ca2+]i
in confluent cells that have been exposed to 5 µM ATP and have been subsequently mechanically stimulated. In response to
ATPo, many cells exhibit
Ca2+ oscillations (Fig. 10,
A-C,
). Because of the variability in sensitivity of individual cells to
ATPo, the single dose of
ATPo evoked differing
Ca2+ responses in adjacent cells.
After mechanical stimulation of a single cell (Fig.
10D, arrow), an intercellular
Ca2+ wave spread through
Ca2+-oscillating and
nonoscillating cells (Fig. 10, E and
F). After passage of the
intercellular Ca2+ wave (Fig. 10,
G-I),
many cells (
) resumed Ca2+
oscillations.

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Fig. 10.
Pseudocolor image of
[Ca2+]i
change in cells exposed to ATPo
and after mechanical stimulation of a single cell (arrows). Cells in a
confluent culture exposed to 5 µM ATP exhibited
Ca2+ oscillations
(A-C,
). Mechanical stimulation of a single cell resulted in passage of an
intercellular Ca2+ wave throughout
field of Ca2+-oscillating and
nonoscillating cells
(D-F).
After passage of Ca2+ wave, cells
continued to exhibit Ca2+
oscillations
(G-I).
White lines, approximate cell outlines. Times are relative to
mechanical stimulation.
|
|
The traces in Fig. 11 show typical
[Ca2+]i
responses of several adjacent cells after application of
ATPo; the variability of the
Ca2+ response is clearly
demonstrated in the traces. Cell B
shows a response typical for a cell with a high sensitivity to
ATPo, whereas
cells A,
C, and
D show responses typical for cells
with an intermediate sensitivity. Although the
Ca2+ response of
cells A,
C, and
D are fundamentally similar, these adjacent cells clearly display
Ca2+ oscillations of different
frequencies and magnitudes, and this emphasizes the asynchronous nature
of adjacent cell Ca2+
oscillations. At 120 s, mechanical stimulation (Fig. 11,
line S) of a single cell
(cell A) initiated the propagation
of an intercellular Ca2+ wave that
spread throughout the cell culture, passing through both
Ca2+-oscillating and
nonoscillating cells. The mechanical stimulation of
cell A resulted in an almost immediate
and sustained
[Ca2+]i
increase in that cell. With a very short time delay (~1 s), the
[Ca2+]i
in cell B rapidly increased and
subsequently decreased to a higher baseline
[Ca2+]i.
In cell C, the
[Ca2+]i
increased after a ~3-s delay to a higher amplitude than the preceding
oscillatory
[Ca2+]i
increases. Interestingly, the next two
Ca2+ oscillations initiated after
a shorter period and at a higher baseline
[Ca2+]i
than did the Ca2+ oscillations
preceding the intracellular wave. In cell
D, after a ~5-s delay, the
[Ca2+]i
increase in the intercellular Ca2+
wave was higher than the initial
[Ca2+]i
increase; however, it failed to reinitiate
Ca2+ oscillations.

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Fig. 11.
Initiation of an intercellular
Ca2+ wave through cells
experiencing ATP-induced Ca2+
oscillations. Outlines (top) and
Ca2+ traces
(bottom) of 4 cells treated with 5 µM ATP are shown. ATPo was
applied to cells (line A) and
elicited characteristic Ca2+
oscillations in the 4 cells. After 120 s, cell
A was mechanically stimulated with a micropipette
(line S), which resulted in an
immediate increase in
[Ca2+]i
of cell A and generation of an
intercellular Ca2+ wave.
Sequential increases in
[Ca2+]i
associated with intercellular Ca2+
wave were observed in cells
B-D, with an
increasing time delay.
[Ca2+]i
increase in cell B was transient and
resulted in a slightly elevated baseline
[Ca2+]i.
In cell C,
[Ca2+]i
increase was followed first by a single
Ca2+ oscillation that had a
shorter period and initiated at a higher
[Ca2+]i
than the previous Ca2+
oscillations and then by resumption of
Ca2+ oscillations similar to those
occurring before Ca2+ wave. In
cell D,
[Ca2+]i
increase was transient and failed to reinitiate
Ca2+ oscillations.
|
|
 |
DISCUSSION |
In this study, we demonstrated by digital imaging fluorescence
microscopy that airway epithelial cells exhibit
Ca2+ oscillations in response to
ATPo. Although
Ca2+ oscillations have been
briefly reported in airway epithelial cells from four species (21, 25,
31, 37), no in-depth characterization has been made of the phenomenon.
Here, we analyzed the mechanism underlying the generation of the
ATP-induced Ca2+ oscillations, the
intercellular heterogeneity of
ATPo responsiveness, and the
intercellular signaling associated with ATP-induced
Ca2+ oscillations.
Signaling pathway involved in the generation of
Ca2+
oscillations.
Airway epithelial cells exhibit a suramin-sensitive
[Ca2+]i
response to ATPo (16), which
suggests that P2 purinogenic receptors are involved in the
[Ca2+]i
response observed in these studies. Airway epithelial cells have been
reported to express several ATP-sensitive purinoceptors, including P2U,
P2T, P2Y (19), and P2X (22). The P2X receptor/channel tentatively
identified in rabbit ciliated epithelium (22) required low (150 µM)
[Ca2+]o
for activity, was not active at high (1.5 mM)
[Ca2+]o,
and was not activated by UTPo
(22). Because our experiments were conducted in ~1.3 mM
[Ca2+]o,
we believe that P2X-receptor activity did not contribute to the
[Ca2+]i
increases observed. UTPo is
ineffective in stimulating P2T and P2X receptors and is much less
potent than ATPo in stimulating P2Y receptors but is of equal or greater potency to
ATPo in stimulating P2U receptors
(12). Consequently, the fact that
UTPo elicited a more robust
[Ca2+]i
response from cells (greater number of cells displaying
Ca2+ oscillations and a greater
number of Ca2+ oscillations per
cell) than ATPo at equimolar
concentrations suggests the involvement of P2U receptors.
Cl
secretion in rat airway
epithelial cells, attributed to apically located P2U receptors,
displayed a similar enhancement by
UTPo (19). In addition, ATP
released from the cystic fibrosis transmembrane conductance regulator
is thought to be involved in the autocrine regulation of
Ca2+-dependent
Cl
channels via apically
located P2U receptors in human airway epithelium cells (10, 42).
The aminosteroid U-73122, a PLC antagonist, prevented an initial
[Ca2+]i
increase in response to ATPo,
consistent with an earlier report suggesting that the
Ca2+ response to
ATPo was mediated by a
PLC/IP3 pathway (17). U-73122, when added to cells experiencing ATP-induced
Ca2+ oscillations, resulted in the
immediate cessation of Ca2+
oscillations. This indicates that both the initial
Ca2+ increase and the subsequent
Ca2+ oscillations utilize common
signaling elements and that Ca2+
oscillations require continuous PLC activity for their maintenance. In
addition, when ATPo is removed
from cells displaying ATP-induced Ca2+ oscillations, the
oscillations immediately cease, suggesting that continuous receptor
signaling is also required for the maintenance of
Ca2+ oscillations. These
experiments, however, cannot distinguish between a cyclic increase in
[IP3]i
produced by the positive feedback of
Ca2+ on
Ca2+-sensitive PLC as proposed by
the cross-coupling model of Ca2+
oscillations (27) and the steady-state increase in
[IP3]i
that drives Ca2+ oscillations as
described in the Ca2+-induced
Ca2+ release model (10). However,
the propagation of intercellular waves in airway epithelial cells does
not appear to involve the regenerative increase in
IP3, which would be expected to
occur if Ca2+-sensitive PLC
isoforms were present in the cell. Hence we believe that the activation
of PLC by ATPo binding to its
receptor results in the steady-state increase in
[IP3]i.
IP3-mediated
Ca2+ oscillations usually rely on
the repetitive release of Ca2+
from internal Ca2+ pools, and when
airway epithelial cells were exposed to
ATPo in nominally
Ca2+-free medium, the cells
displayed characteristic Ca2+
oscillations for a number of minutes. These results indicate a
dependence on internal Ca2+ stores
for the oscillations. Refilling of the internal
Ca2+ stores is mediated by the
action of Ca2+-ATPases, which can
be inhibited by thapsigargin. Airway epithelial cells treated for 60 s
with thapsigargin showed a slight increase in the
[Ca2+]i
during thapsigargin treatment; however, when
ATPo was applied, the cells
responded with a large initial
[Ca2+]i
increase that remained elevated and failed to exhibit
Ca2+ oscillations. Thus no
ATP-induced Ca2+ oscillations were
observed after discharge of the internal pools when the pool was not
allowed to refill. Furthermore, by applying thapsigargin 60 s after the
addition of ATP and the onset of
Ca2+ oscillations, the amplitude
of the Ca2+ oscillations was
progressively diminished and the baseline
[Ca2+]i
was increased until the Ca2+
oscillations ceased at an elevated
[Ca2+]i.
A dependence on thapsigargin-sensitive internal
Ca2+ pools is a similar
requirement for the ATP-induced
Ca2+ oscillations of chondrocytes
(8) and bile duct epithelial cells (29).
Characteristics of
Ca2+
oscillations.
Four basic responses to ATPo were
identified in this study. These were
1) a single
Ca2+ increase after
ATPo stimulation;
2) the initiation of a few irregular Ca2+ oscillations;
3) the initiation of regular,
periodic Ca2+ oscillations; and
4) a sustained elevation in the
baseline
[Ca2+]i
after an initial Ca2+ increase.
The heterogeneity of the Ca2+
response in airway epithelial cells was very similar to the
heterogeneous Ca2+ responses to
ATPo reported in glial cells (49),
bile duct cells (29), megakaryocytes (46), and chondrocytes (8). In
airway epithelial cells, however, these four
Ca2+ responses were reproduced in
individual cells exposed to different [ATP]o values.
Therefore, the heterogeneity of the
Ca2+ response was due in part to a
100-fold difference in the sensitivity of individual cells to
ATPo and allowed us to
characterize individual cells as having low, intermediate, and high
sensitivities to ATPo. The basis
of this differential sensitivity was not determined but may arise from
variations in the expression, density, or sensitivity of the ATP
receptor (P2U), IP3Rs, or other
components of the signaling pathway. Although these differences may
result in different resting [Ca2+]i
values, a correlation between ATP sensitivity and resting
[Ca2+]i
was not found. This suggests that the ATP sensitivity may only be
manifested after stimulation.
Ca2+-signaling differences may
also arise from species-specific differences. Although
Ca2+ oscillations have been
observed in airway epithelial cells from rabbit, cow (21), mouse
(Evans, unpublished observations), and sheep (37), there has been no
report of Ca2+ oscillations in
human airway cells except for serous gland cells (25). In view of the
widespread occurrence of Ca2+
oscillations, it would be surprising to find that human cells fail to
demonstrate Ca2+ oscillations.
Differences in the signaling components and the
Ca2+ response of cells can also
arise from culturing conditions such as variations in the composition
of the matrix and medium, variations in the Ca2+-signaling phenotype between
normal and transformed cell lines, and the applied agonist
concentrations. In addition,
[Ca2+]i
measurements from populations of cells rather than from individual cells would fail to reveal heterogeneous
Ca2+ oscillations such as those
reported here. These reasons may also account for why the observation
of Ca2+ oscillations in airway
epithelial cells has varied among investigators.
If the steady-state
[IP3]i
is controlled by the degree of receptor activation, as it is in other
cell types (4), then the four basic
Ca2+ responses observed in airway
epithelial cells reflect the changes in
[IP3]i,
a hypothesis that is consistent with the mathematical modeling of
Ca2+ oscillations (43). When the
[ATP]o was relatively
low (relative to the intrinsic sensitivity of the cell to
ATPo), reflecting relatively low
receptor activation and a low steady-state
[IP3]i, the cell was unable to support
Ca2+ oscillations and responded to
ATPo with a single
[Ca2+]i
increase. In the
[ATP]o range that
yielded Ca2+ oscillations, the
Ca2+ oscillation frequency
increased with increased
[ATP]o, an observation common to many cell types stimulated by a wide variety of agonists such
as vasopressin (33), phenylephrine (34), acetylcholine (29), and ATP
(8) and possibly reflected increased
[IP3]i. In some experiments, the first few
Ca2+ oscillations had shorter
periods and initiated at a higher
[Ca2+]i
than subsequent Ca2+ oscillations,
which may be due to an initial
[IP3]i
spike that declines to a steady-state
[IP3]i.
Exhibition of preceding higher-frequency Ca2+ oscillations initiating at
higher
[Ca2+]i
values is a feature common in cells to many agonists including ATP (8,
29, 46). At relatively high
[ATP]o values, when the steady-state
[IP3]i
would be high, the cells responded with a sustained increase in
[Ca2+]i
and no Ca2+ oscillations.
Spatial characteristics of
Ca2+
oscillations.
The pattern of ATP-induced Ca2+
oscillations was intrinsic to each cell, and
Ca2+ oscillations within one cell
failed to influence the
[Ca2+]i
activity of adjoining cells in a manner similar to the asynchronous ATP-induced Ca2+ oscillations that
have been observed in bile duct cells (29) and MDCK cells (35) and are
consistent with the behavior of spontaneous
Ca2+ oscillations in airway
epithelial cells (6). After a nearly synchronous increase in
[Ca2+]i
in the cells in response to the addition of
ATPo, each cell displayed a unique
Ca2+ response. In contrast,
ATP-induced Ca2+ oscillations
initiate intercellular Ca2+ waves
in cultures of chondrocytes (8), and a variety of agonists generate
Ca2+ oscillations that spread as
intercellular Ca2+ waves in liver
tissue (28, 33) and hepatocytes (48). Interestingly, in MDCK cells,
bradykinin and thrombin, but not
ATPo, stimulate synchronized
Ca2+ oscillations, suggesting that
the synchrony of Ca2+ oscillations
in adjoining cells may be a function of the agonist (35). The synchrony
of Ca2+ oscillations in MDCK cells
and the spreading of intercellular Ca2+ waves from
Ca2+ oscillations in hepatocytes
and salivary glands, however, are dependent on gap junction
communication. Disruption of communication by octanol (35) or
-glycyrrhetinic acid (48) leads to asynchronous Ca2+ oscillations or the failure
of Ca2+ oscillations to initiate
intercellular Ca2+ waves without
affecting Ca2+ oscillations
themselves, suggesting the gap junction communication of a signaling
molecule between cells to maintain synchrony.
Intercellular communication during
Ca2+
oscillations.
In airway epithelial cells, mechanical stimulation of one cell results
in propagated increases in
[Ca2+]i
to neighboring cells or an intercellular
Ca2+ wave. Passage of an
intercellular Ca2+ wave in airway
epithelial cells is mediated by the gap junction diffusion of
IP3 generated in the stimulated
cell (6). Consequently, the passage of an intercellular
Ca2+ wave through a field of
ATP-stimulated cells, some of which were displaying
Ca2+ oscillations, suggests that
the inability of the
Ca2+-oscillatory behavior of one
cell to influence a neighboring cell is not due to the disruption of
gap junction communication between cells. This inability instead
demonstrates that Ca2+ alone is
insufficient as an intercellular messenger to communicate Ca2+ changes to neighboring airway
epithelial cells even when the IP3Rs of the neighboring cells are
sensitized to IP3, although Ca2+ may act as the messenger in
other cell types (28, 33, 48). Indeed, the magnitude of the
Ca2+ increase associated with the
Ca2+ oscillations in the cells is
not different from the magnitude of the
[Ca2+]i
change associated with the passage of the intercellular
Ca2+ wave, thus demonstrating that
the
[Ca2+]i
increase associated with the wave cannot account for
Ca2+ wave transmission.
Although it has been reported that mechanical stimulation of airway
epithelial cells results in the release of ATP (14), we do not believe
that the intercellular propagation of
Ca2+ waves described above relies
on this mechanism. We repeatedly failed to observe
Ca2+ oscillations in cells after
the passage of a mechanically stimulated intercellular
Ca2+ wave, which we would expect
if Ca2+ waves were dependent on
ATPo. Also, although the
Ca2+ response in airway epithelial
cells is blocked by suramin, a P2-receptor antagonist, the propagation
of mechanically stimulated intercellular
Ca2+ waves is not (17). Moreover,
fluid flow over cells during mechanical stimulation fails to bias the
direction of spread of the subsequent intercellular
Ca2+ waves (17).
Associated with the passage of the intercellular
Ca2+ wave through ATP-induced
Ca2+-oscillating cells was a
transient increase in the oscillation frequency and the initiation of
Ca2+ oscillations at a higher
[Ca2+]i.
As discussed above, this observation is consistent with a transient
increase in
[IP3]i
over the steady-state
[IP3]i,
which would be expected because
IP3 diffuses outward from the
stimulated cell ahead of the Ca2+
wave and then is metabolized and diffused into the surrounding cells.
Although IP3 can apparently
traverse the gap junctions between neighboring cells stimulated with
ATPo, we suggest that no
IP3 gradient is established
between ATP-stimulated cells, so no intercellular
Ca2+ waves are initiated from any cell.
In summary, two separate Ca2+
signals can occur in airway epithelial cells:
Ca2+ oscillations that are
intrinsic and confined to an individual cell and intercellular
Ca2+ waves that are generated at a
distance and encompass a number of cells. We show that the two
Ca2+ signals can occur
simultaneously within cells and suggest that Ca2+ oscillations may serve to
regulate individual cell functions, whereas intercellular
Ca2+ waves coordinate cooperative
cell activity.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood Grant
HL-49288 (to M. J. Sanderson).
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. H. Evans,
Dept. of Physiology, S4-315, Univ. of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655 (E-mail:
John.Evans{at}ummed.edu).
Received 5 November 1998; accepted in final form 8 March 1999.
 |
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