Effect of FK506 on ATP-induced intracellular calcium
oscillations in cow tracheal epithelium
Soichiro
Kanoh1,
Mitsuko
Kondo2,
Jun
Tamaoki2,
Hideki
Shirakawa3,
Kazutetsu
Aoshiba2,
Shunichi
Miyazaki3,
Hideo
Kobayashi1,
Naokazu
Nagata1, and
Atsushi
Nagai2
1 Third Department of Medicine,
National Defense Medical College, Saitama 359; and
2 First Department of Medicine and
3 Department of Physiology,
Tokyo Women's Medical College, Tokyo 162, Japan
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ABSTRACT |
To elucidate the effect of FK506 on
Ca2+ oscillations in airway
epithelium, we investigated cultured cow tracheal epithelial cells with
a Ca2+ image-analysis system. ATP
(1 µM) induced long-lasting
Ca2+ oscillations, having nearly
constant peak values (300-400 nM) and intervals (20-40 s) in
subconfluent cells but not in confluent cells. These responses were
gradually attenuated and abolished by the addition of FK506. Rapamycin,
which binds the FK506-binding protein (FKBP), likewise inhibited
Ca2+ oscillations, whereas
cyclosporin A, a calcineurin inhibitor, did not. Treatment of cells
with FK506 decreased Ca2+ content
in thapsigargin-sensitive stores, suggesting that the partial depletion
of the stores causes the inhibition of
Ca2+ oscillations.
Immunocytochemistry revealed the existence of cytoplasmic FKBP-like
immunoreactivities. The expression of a 12-kDa FKBP was greater in
subconfluent cells than in confluent cells as determined by Western
blotting, suggesting that the 12-kDa FKBP may be one of the factors
that regulates Ca2+ oscillations.
Therefore, FK506 possesses an inhibitory action on the
Ca2+ response via intracellular
FKBP but not via calcineurin, which may result in modification of
airway epithelial functions.
airway epithelium; adenosine 5'-triphosphate; FK506-binding
protein; rapamycin; cyclosporin
 |
INTRODUCTION |
IN AIRWAY EPITHELIUM, several inflammatory mediators
such as bradykinin and ATP induce an increase in intracellular free
Ca2+ concentration
([Ca2+]i)
(19, 22, 24), which mediates various cell functions including ion
transport (8, 19) and mucus secretion (14). Repetitive spikes of
[Ca2+]i
(Ca2+ oscillations) are observed
during agonist stimulation in a wide variety of electrically
nonexcitable and excitable cells and are considered as physiologically
significant Ca2+ kinetics that
regulate cellular functions (2, 11, 20). Airway epithelial cells have
been reported to show Ca2+
oscillations in response to mechanical stimulation (4), acetylcholine (25), and neutrophil elastase (18). Although the mechanism of
Ca2+ oscillations is not fully
understood, the intracellular Ca2+
release channel (CRC) of the endoplasmic reticulum (ER) such as the
inositol 1,4,5-trisphosphate
(IP3) receptor
(IP3R) plays a central role (2, 4,
11, 20).
FK506, a macrolide immunosuppressant drug, binds the FK506-binding
protein (FKBP), and the FK506-FKBP complex inhibits the Ca2+-dependent phosphatase
calcineurin, thereby preventing calcineurin-dependent interleukin-2
transcription and T-cell proliferation (17, 26). The 12-kDa (FKBP12)
and 12.6-kDa FKBPs are associated with the ryanodine receptor (RyR) of
the sarcoplasmic reticulum in skeletal (12) and cardiac muscle (29)
cells, respectively. FK506 causes dissociation of FKBP from RyR and
alters its CRC function (3). Likewise, recent evidence suggests that
FKBP12 is also associated with the
IP3R and that disrupting this
complex by FK506 results in alternation of CRC conductance (6).
However, the effect of FK506 on
Ca2+ oscillations remains unknown.
In the present study, we demonstrate Ca2+ oscillations induced by
exogenous ATP and its inhibition by FK506 in fura 2-loaded cow tracheal
epithelial cells.
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MATERIALS AND METHODS |
Cell culture. Cow tracheae were
obtained from a slaughterhouse, and tracheal epithelial cells were
isolated by protease as previously described (15). Briefly, strips of
epithelium were pulled off the submucosa, washed four times with
phosphate-buffered saline (PBS) containing 5 mM dithiothreitol, and
rinsed two times with PBS. Epithelial tissues were digested with PBS
containing 0.05% protease at 4°C overnight. After neutralization
of the protease with 5% fetal calf serum (FCS), the cells were
pelleted (200 g for 10 min) and
suspended in 50% Dulbecco's modified Eagle's medium (DMEM) and 50%
Ham's F-12 nutrient mixture that contained 5% FCS, 1% nonessential
amino acids, 100 U/ml of penicillin, 100 µg/ml of
streptomycin, and 50 µg/ml of gentamicin. The isolated cells were
plated at a density of 1.0 × 105
cells/cm2 on a glass-bottomed
petri dish (MatTek, Ashland, MA) coated with human placental collagen.
The medium was changed every 2 days. The cells were cultured for
3-5 or 7-8 days to a subconfluent or confluent stage,
respectively, to observe the difference in ATP-induced
Ca2+ responses between these
different culture conditions.
Measurement of
[Ca2+]i
in single cells.
The dish on which the cells were grown was washed with Hanks' balanced
salt solution (HBSS) that contained 10 mM HEPES, pH 7.4, and was loaded
with 10 µM fura 2-AM for 1 h at 37°C. The dish was then washed
several times with HEPES-buffered HBSS and mounted on the stage of an
inverted microscope (Diaphot 300, Nikon, Tokyo, Japan). The temperature
was kept at 37°C by a ring heater surrounding the dish. For
excitation of fura 2 fluorescence, ultraviolet (UV) light of 340- or
380-nm wavelength was produced by a xenon lamp and narrow band-pass
filters and applied to the cells through a ×40 objective lens
(Fluor 40, Nikon). Emission fluorescence (F) was led to a
silicon-intensifier target camera through a 510 ± 10-nm band-pass
filter. Ca2+ images of the cells
were obtained at 3- or 4-s intervals unless otherwise indicated by
alternately applying 340- and 380-nm UV light for 0.125 s (four video
frames) for each. Data sets were stored on the hard disk of the
computer as eight-bit digital images (256 × 256 pixels) and
processed to calculate the ratio of 340- to 380-nm fluorescence later.
The averaged values of the ratios in individual cells were obtained in
an optical field in which ~40 cells were sampled simultaneously. A
calibration curve between the ratio and
[Ca2+]i
was obtained by measuring the ratios of
Ca2+-N-(2-hydroxyethyl)EDTA
buffer solutions. All these procedures were performed with an
image processor (Argus-50/CA system, Hamamatsu Photonics,
Hamamatsu, Japan) (10).
Electron microscopy. To examine
morphological differences between subconfluent and confluent cells, the
cells on glass-bottomed petri dishes were fixed in 2.5% glutaraldehyde
for 2 h and postfixed in 1% osmium tetroxide for 1 h. After
dehydration in a graded series of alcohols, the cells were embedded in
Epon. Thin sections (80 nm), which were cut perpendicular to the plane
of the cell sheet, were mounted on copper grids, stained with lead
citrate and uranyl acetate, and examined with a transmission electron microscope (H-7000, Hitachi, Tokyo, Japan).
Immunocytochemistry for FKBP12.
Indirect immunofluorescence was used to detect FKBP12 in the cultured
tracheal epithelial cells that had been fixed in 3% paraformaldehyde
and permeabilized with 0.5% Triton X-100. The cells were incubated
with a 1:10 dilution of monoclonal anti-FKBP12 antibody (2C1) for 30 min at room temperature. After the antibody was removed, the cells were
washed three times for 10 min each with PBS. Then the cells were
incubated with a 1:100 dilution of the FITC-conjugated anti-mouse IgG
goat antibody for 1 h at room temperature, washed with PBS, and
observed under a fluorescence microscope. As negative controls, PBS was
used as the first antibody instead of the anti-FKBP12 antibody to
evaluate nonspecific staining.
Immunoblotting for FKBP12. Western
blot analysis was used to assess a possible difference in the amount of
FKBP12 between different culture conditions. Subconfluent (4-day
culture) and confluent (8-day culture) cells plated on collagen-coated
dishes were scraped and homogenized in radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris · HCl, 0.5% Nonidet
P-40, 0.1% SDS, and 2 mM EDTA, pH 7.4) containing 10 µg/ml each of
aprotinin, leupeptin, and phenylmethylsulfonyl fluoride followed by
centrifugation (12,000 g for 30 min)
at 4°C. Equal amounts of protein (75 µg/lane) from the
supernatants were separated by 15% SDS-PAGE followed by
electrophoretic transfer to polyvinylidene difluoride membrane. Recombinant human FKBP12 (0.3 µg) was also loaded on the gel as a
positive control. The membrane was blocked with blocking buffer (150 mM
NaCl, 50 mM Tris, and 0.1% Tween 20, pH 7.5) containing 5% skimmed
milk and 1% goat serum at 4°C overnight. Subsequently, the
membrane was incubated with a 1:200 dilution of monoclonal anti-FKBP12
antibody (2C1) for 1 h at room temperature. As a negative control, a
piece of membrane was cut along the interlane and incubated with
blocking buffer instead of the anti-FKBP12 antibody. The membranes were
then incubated with a 1:2,500 dilution of peroxidase-conjugated anti-mouse IgG goat antibody for 1 h at room temperature. The blots
were developed with an enhanced chemiluminescent substrate (Pierce,
Rockford, IL). Protein band densities were measured with a densitometer
(ATTO densitograph, Atto, Tokyo, Japan), and the values are expressed
in arbitrary optical density units.
Drugs. DMEM, Ham's F-12 medium, and
nonessential amino acids were purchased from GIBCO BRL (Tokyo, Japan).
Fura 2-AM was obtained from Dojindo Laboratories (Kumamoto, Japan). All
other chemicals, FITC-conjugated or peroxidase-conjugated anti-mouse
IgG goat antibody, and recombinant human FKBP12 were obtained from
Sigma (St. Louis, MO). FK506 and anti-FKBP12 antibody (2C1) were gifts
from Fujisawa Pharmaceutical (Osaka, Japan). FK506, rapamycin, and
cyclosporin A were dissolved in ethanol and used at a final ethanol
concentration of <0.1%, and 0.1% ethanol-HEPES-buffered HBSS was
employed as a vehicle control.
Statistics. Data are expressed as
means ± SE. Statistical analysis was performed by two-tailed paired
or unpaired Student's t-test, and a
P value of <0.05 was considered significant.
 |
RESULTS |
ATP-induced
Ca2+ responses.
At the subconfluent stage in culture, the mean resting
[Ca2+]i
in single epithelial cells from cow tracheae was 123.1 ± 1.5 nM
(n = 1,881 cells). After
stimulation with 100 µM ATP, >95% of the cells showed a rapid
elevation in
[Ca2+]i.
This Ca2+ response was biphasic,
consisting of an initial transient rise and a following sustained
elevation (Fig.
1A).
When the concentrations of ATP were decreased to 1-10 µM, some
cells showed repetitive Ca2+
spikes (i.e., Ca2+ oscillations).
These Ca2+ oscillations could be
divided into two groups with respect to their response patterns. One
was a decaying pattern in which
Ca2+ oscillations, consisting of
at least three obvious
[Ca2+]i
peaks, were gradually attenuated in amplitude and frequency and
abolished within 5 min (Fig. 1B).
The other was a long-lasting pattern (Fig.
1C). This oscillatory response
showed discrete Ca2+ spikes
arising from a steady
[Ca2+]i
level, namely a transient pattern of
Ca2+ oscillations. These
Ca2+ oscillations had almost
constant intervals (20-40 s) and peak values (300-400 nM) in
individual cells and lasted for at least 20-30 min.

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Fig. 1.
Representative recordings of ATP-induced
Ca2+ responses in single cow
tracheal epithelium under subconfluent culture condition.
A: 100 µM ATP induced a transient
rapid increase in intracellular
Ca2+ concentration
([Ca2+]i)
followed by a sustained elevation.
B: decaying
Ca2+ oscillations induced by 10 µM ATP in which oscillations were gradually attenuated in amplitude
and frequency and abolished within 5 min.
C: long-lasting
Ca2+ oscillations induced by 1 µM ATP. In this pattern, repetitive spikes of
[Ca2+]i
lasted for at least 20-30 min. Arrows, addition of ATP.
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The Ca2+-response data for the
individual cells under subconfluent culture conditions are summarized
in Fig.
2A. In
subconfluent cells, the percentage of cells that showed a decaying
pattern and a long-lasting pattern of
Ca2+ oscillations was ~10 and
30% at optimal ATP concentrations of 10 and 1 µM, respectively. In
contrast, the percentage of cells that showed a transient
[Ca2+]i
response was increased as the concentration of ATP increased. Similar
experiments were performed in confluent epithelial cells (Fig.
2B). Under this condition, the mean
resting
[Ca2+]i
was 116.7 ± 2.4 nM (n = 782 cells), and the percentage of cells that showed a transient
[Ca2+]i
response was increased in proportion to the concentration of ATP as
with subconfluent cells. However, long-lasting
Ca2+ oscillations were rarely
observed and the percentage was <1%, whereas decaying
Ca2+ oscillations were recognized
in ~25% of cells at 10 µM ATP.

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Fig. 2.
Relationship between ATP concentration ([ATP]) and
percentage of responding cells that showed transient
Ca2+ rise (open bars), decaying
Ca2+ oscillations (hatched bars),
and long-lasting Ca2+ oscillations
(solid bars) under different culture conditions. Data are means ± SE; n = 4-20 experiments/ATP
concentration where 30-40 cells were observed in individual
experiments. A: under subconfluent
culture conditions, nearly all cells showed transient
Ca2+ rise in response to >100
µM ATP, whereas greatest percentage of long-lasting oscillating cells
was observed at 1 µM ATP. B: under
confluent culture conditions, long-lasting
Ca2+ oscillations were rarely
observed and percentage was <1%, although decaying
Ca2+ oscillations were recognized
in ~25% cells at 10 µM ATP.
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Morphological examination. Because an
apparent morphological difference was not observed with light
microscopy between cells that showed oscillatory and nonoscillatory
Ca2+ responses, ultrastructural
examination was performed with electron microscopy. As shown in Fig.
3, subconfluent cells cultured for 4 days
had microvilli and tight junctions, which are typical characteristics of epithelial cells, although they were flattened in appearance. Similarly, confluent cells cultured for 8 days had slightly longer and
more apparent microvilli in addition to the above findings. However,
both cultured cells lacked cilia and secretory granules and showed an
undifferentiated appearance.

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Fig. 3.
Transmission electron micrographs of cow tracheal epithelial cells
cultured for 4 [A and
B (enlargement of box in
A)] and 8 days
[C and
D (enlargement of box in
C)]. Both cultured cells had
typical epithelial characteristics such as microvilli and junctional
complexes (arrows) but lacked cilia and secretory granules. Bars: 5 µm in A and
C; 500 nm in
B and
D.
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Effect of FK506 on ATP-induced
Ca2+
oscillations.
Cultured airway epithelial cells showed several patterns of
Ca2+ responses according to ATP
concentrations and culture conditions. In this experiment, we selected
1 µM ATP and subconfluent cells to examine the effect of FK506 on
Ca2+ oscillations because this ATP
concentration and culture condition seemed optimal to induce
long-lasting Ca2+ oscillations.
Vehicle alone added during Ca2+
oscillations had no effect on ongoing
Ca2+ spikes (data not shown).
After the addition of 1 µM FK506 during Ca2+ oscillations, the first
Ca2+ spike became slightly larger,
but the subsequent Ca2+
oscillations were gradually attenuated in amplitude and frequency and
were eventually abolished (Fig.
4A).
Likewise, the addition of 10 µM FK506 produced a more pronounced
inhibition of ongoing Ca2+
oscillations (Fig. 4B). This
inhibitory effect was prevented when excess recombinant human FKBP12
was added to the extracellular milieu before FK506 (Fig.
4C). Rapamycin (1 µM), another
immunosuppresant drug that binds FKBP with high affinity, showed a
similar inhibitory effect on ongoing
Ca2+ oscillations as FK506 (Fig.
4D), whereas cyclosporin A (1 µM), a specific calcineurin inhibitor that does not bind FKBP, had little
effect (data not shown).

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Fig. 4.
Effects of FK506, rapamycin, and thapsigargin (TG) on ATP-induced
Ca2+ oscillations.
A: 1 µM FK506 reduced amplitude and
frequency of Ca2+ oscillations
that were eventually abolished. B: 10 µM FK506 strongly inhibited Ca2+
oscillations. C: 12-kDa FKBP (FKBP12)
per se did not affect Ca2+
response but prevented inhibitory effect of FK506 on
Ca2+ oscillations.
D: after addition of 1 µM rapamycin,
Ca2+ oscillations declined as with
FK506. E: 3 µM TG caused an increase
in
[Ca2+]i
and then abolished Ca2+
oscillations. Arrows, addition of drug.
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Effect of FK506 on
Ca2+ content in
thapsigargin-sensitive stores.
Thapsigargin (TG), a specific ER
Ca2+-pump inhibitor (27), added
during Ca2+ oscillations caused a
sustained Ca2+ release and
thereafter abolished the oscillations (Fig.
4E). Moreover, as shown in Fig.
5A, the
addition of TG evoked a rise in
[Ca2+]i,
an effect that may be due to the depletion of intracellular Ca2+ stores, and inhibited the
subsequent elevation in ATP (100 µM)-induced [Ca2+]i,
implying that the response to ATP is attributable to mobilization of
Ca2+ from TG-sensitive stores. We
thus examined the effect of FK506 on
Ca2+ content in TG-sensitive
stores to elucidate the site of action of FK506. To do so, EGTA
(5 mM) was added to the medium to chelate extracellular
Ca2+ 30 s before the addition of
TG because depletion of Ca2+ in ER
causes Ca2+ influx from the
external solution through the capacitative
Ca2+ entry pathway (23), and the
area beneath the records of the rise in TG-evoked
[Ca2+]i
above the basal level was integrated. Examples of estimated Ca2+ content in TG-sensitive
stores treated with vehicle alone and with 1 µM FK506 for 5 min are
demonstrated in Fig. 5, B and
C, insets, respectively. Similar
experiments were carried out at various time points (1, 3, 5, and 10 min) after the addition of vehicle alone or FK506, and the results were
compared with those in the cells with no drugs added (control; 100%).
As shown in Fig. 6, vehicle alone had no
effect, but FK506 (1 µM) significantly diminished the ER
Ca2+ content to 69.6 ± 6.3 and
70.0 ± 6.5% at 5 and 10 min, respectively, after the addition
(P < 0.01;
n = 60 cells/group).

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Fig. 5.
Inhibition of ATP-induced increase in
[Ca2+]i
by TG and effect of FK506 on TG-evoked
[Ca2+]i
rise. A: TG evoked a transient
[Ca2+]i
rise followed by a sustained response. Subsequent addition of ATP did
not cause a substantial increase in
[Ca2+]i.
After treatment with vehicle alone
(B) or FK506
(C) for 5 min, TG was added in
presence of EGTA.
[Ca2+]i
showed transient increase and then declined to below basal level. As an
index of Ca2+ content in
TG-sensitive stores, area beneath records of TG-evoked
[Ca2+]i
rise above basal level was integrated
(insets, solid areas).
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Fig. 6.
Effect of FK506 on Ca2+ content in
TG-sensitive stores. Cells were pretreated with vehicle alone ( ) or
FK506 (1 µM; ) for indicated times, and
Ca2+ content in TG-sensitive
stores was determined. Values are means ± SE of percentage of
control response (no drug added); n = 60 cells/time point. Vehicle alone had no effect, whereas FK506
significantly diminished Ca2+
content 5 min later. Significantly different
(P < 0.01) from: ** control
value;  corresponding response to vehicle alone.
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Effect of FK506 on basal
[Ca2+]i
level.
To determine the effect of FK506 itself on basal
[Ca2+]i
level, 1 µM FK506 was added to cultured airway epithelial cells and the response was compared with that of the vehicle control.
Ca2+ images were acquired every
minute to avoid the influence of UV light for excitation of the cells
and fura 2. Basal
[Ca2+]i
before the addition of FK506 was 124.3 ± 3.1 nM
(n = 120 cells) and was significantly
elevated 3 min after the addition (125.2 ± 3.2 nM;
P < 0.05). The increase in
[Ca2+]i
5 min after FK506 (130.5 ± 3.3 nM;
P < 0.001) was >5 nM, and it
reached a plateau thereafter, whereas vehicle alone had no such effect
(Fig. 7).

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Fig. 7.
Effect of FK506 on basal
[Ca2+]i
level in airway epithelial cells. Vehicle alone ( ) or FK506 (1 µM;
) was added, and
[Ca2+]i
was measured every minute. Values are means ± SE;
n = 120 cells/time point. Basal
[Ca2+]i
after addition of FK506 was significantly elevated 3 min later and
reached a plateau at 5 min, whereas vehicle had no effect on basal
[Ca2+]i.
Significantly different from initial (0-min) baseline
[Ca2+]i
value: * P < 0.05;
*** P < 0.001.
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Effect of FK506 on ATP-induced transient
Ca2+ rise.
To examine whether the reduction in
Ca2+ content in TG-sensitive
stores by FK506 has an effect on the
Ca2+ response induced by a high
concentration of ATP, 100 µM ATP was added after treatment with 1 µM FK506 or vehicle alone for 10 min. As shown in Fig.
8, the peak
[Ca2+]i
level was smaller in FK506-treated cells than in vehicle-treated cells.
The increases in
[Ca2+]i
from the basal level were 494.4 ± 12.4 and 647.2 ± 18.8 nM, respectively (P < 0.001;
n = 120 cells/group).

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Fig. 8.
Representative recordings of ATP-induced transient
Ca2+ rise in cultured cow tracheal
epithelium treated with FK506 (solid line) and vehicle alone (dotted
line). After treatment with 1 µM FK506 or vehicle alone for 10 min,
ATP was added (arrow).
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Detection of FKBP12. To assess whether
FKBP12 is present in our cultured tracheal epithelial cells,
immunocytochemical staining with a monoclonal anti-FKBP12 antibody was
performed. As shown in Fig. 9,
immunofluorescence was observed in the cytoplasm of subconfluent cells,
indicating that epithelial cells possess FKBP-like immunoreactivities.
The localization of FKBP12 was likewise found in confluent cells (data
not shown). To further investigate whether the amount of FKBP12 is
different according to culture periods, a Western blotting technique
was used. As shown in Fig.
10A,
specific binding for FKBP12 was observed in both subconfluent and
confluent cells (lanes 2 and
3, respectively), and densitometric
estimation of the FKBP12 quantity revealed that subconfluent cells had
more FKBP-like immunoreactivities than did confluent cells (Fig.
10B).

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Fig. 9.
Indirect immunofluorescence of FKBP12 in cow cultured tracheal
epithelium at subconfluent stage in culture. Immunocytochemical
staining was performed with (B) and
without (A) anti-FKBP12 antibody as
a first antibody. Original magnification, ×400.
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Fig. 10.
Expression of FKBP12 in subconfluent and confluent cells as determined
by Western blot analysis. A:
recombinant human FKBP12 (0.3 µg; lane
1) and equal amounts of extracted protein (75 µg)
from subconfluent (lanes 2 and
4) and confluent
(lane 3) cells were subjected to
15% SDS-PAGE and electroblotted onto polyvinylidene difluoride
membrane, which was developed with (lanes
1-3) and
without (lane 4) anti-FKBP12
antibody and peroxidase-conjugated anti-mouse IgG goat antibody. Blots
were visualized with enhanced chemiluminescence.
B: FKBP12 band densities of
subconfluent and confluent cells were measured with a densitometer.
Results are representatives of 3 independent experiments.
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 |
DISCUSSION |
This study showed that low concentrations of ATP induce long-lasting
Ca2+ oscillations in subconfluent
cells and that FK506 inhibits Ca2+
oscillations in a dose-dependent manner. It is well known that inflammatory mediators such as bradykinin and ATP evoke an increase in
[Ca2+]i
(19, 22, 24), but there are few reports regarding
Ca2+ oscillations induced by
mediators in airway epithelium (18, 23). The range of ATP
concentrations that evoked oscillatory responses was limited, and
>100 µM ATP did not cause Ca2+
oscillations in most cells. We thus speculate that moderate stimulation might be appropriate for ongoing
Ca2+ oscillations in individual
cells. These observations are consistent with previous reports (28, 32)
using different agonists on other cell types. In addition, even at an
optimal concentration of ATP (1 µM), only 30% of the cells at the
subconfluent stage showed long-lasting
Ca2+ oscillations. However, we
could not find the morphological difference with light microscopy
between the cells that showed Ca2+
oscillations and those that did not (data not shown). Moreover, there
was no difference in the ultrastructure between subconfluent that
tended to show oscillatory responses and confluent cells that did not
(Fig. 3). Regarding the difference in the incidence of agonist-induced
Ca2+ oscillations according to
different culture periods, Salathe and Bookman (25) showed that
acetylcholine produced Ca2+
oscillations in sheep tracheal epithelial ciliated cells only early in
culture (1-3 days). Therefore, the cells cultured for relatively
short periods may have the ability to produce long-lasting Ca2+ oscillations. Although the
reason for this is unknown, the difference in the amount of FKBP12
could be involved, as described later.
Extracellular ATP stimulates P2U receptor in the airway epithelium,
activates phospholipase C, and produces
IP3, which in turn mobilizes
Ca2+ from the ER via the
IP3R (14). Intracellular
Ca2+ pools play a role in the
regulation of
[Ca2+]i
and are profoundly involved in
Ca2+ oscillations (2, 4, 11, 20).
Some investigators (1, 3, 7, 13) have shown that FKBPs associate with
both skeletal and cardiac RyRs and modulate CRC function in lipid
bilayers and that FK506 or rapamycin increases the open probability of
the RyRs. Furthermore, Cameron et al. (6) demonstrated that the physiological role of FKBP is to stabilize the CRC function of IP3R as well as of RyR by
promoting optimal cooperativity among four subunits of the channel and
that dissociation of FKBP12 from the
IP3R by FK506 and rapamycin causes
leakiness in the gating properties of the channel. We therefore
expected that FK506 added during
Ca2+ oscillations would potentiate
Ca2+ release. It is of interest,
however, that FK506 gradually attenuated and abolished ATP-induced
Ca2+ oscillations, although the
first Ca2+ spike was potentiated
after the addition of FK506. One possible explanation for this
observation would be that the slightly larger Ca2+ spike reflects the increased
leakiness of CRC, and the following suppression of
Ca2+ oscillations may be exerted
by the decrease in the net Ca2+
accumulation of intracellular Ca2+
stores. To confirm this, we examined the
Ca2+ content in TG-sensitive
stores in FK506-treated cells because the source of ATP-stimulated
Ca2+ release appeared to be
TG-sensitive stores (Figs. 4E and
5A). In the presence of EGTA, the
rise in
[Ca2+]i
induced by TG is supposed to reflect the release from intracellular Ca2+ stores (Fig.
5B). Under this condition, 1 µM
FK506 significantly decreased the ER
Ca2+ content by 30% 5 min after
being added (Figs. 5C and 6). If this partial depletion of ER Ca2+
content is due to the increased leakiness of CRC, it must be preceded
by the elevation in
[Ca2+]i
resulting from leaked Ca2+. This
hypothesis was supported by the finding that basal
[Ca2+]i
was significantly elevated 3 min after the addition of 1 µM FK506 and
formed a new
[Ca2+]i
level that was 5 nM higher than the initial level in 5 min (Fig. 7).
These results are in accord with previous reports that the leakiness of
CRC by FK506 or rapamycin causes partial depletion of
Ca2+ in RyR-gated (3, 30, 31) or
IP3R-gated (6) stores. However,
there seems to be a discrepancy in the time course between the
depletion of Ca2+ content and the
immediate inhibition of Ca2+
oscillations by FK506. Brillantes et al. (3) have shown that when
caffeine was applied successively to rapamycin-treated sarcoplasmic reticulum vesicles from rabbit skeletal muscle,
Ca2+ release induced by the second
caffeine addition was smaller than that by the first, indicating that
the vesicles are partially depleted of
Ca2+. Timerman et al. (30) also
reported that the time to load
Ca2+ into terminal cisternae
vesicles treated with FK506 was distinctly prolonged and that their
Ca2+ uptake rate was reduced
because of the leakiness of RyRs. Taken together, the immediate
inhibition of Ca2+ oscillations by
FK506 may be due to insufficient time to take up
Ca2+ fully into the ER. Because
repetitive Ca2+ spikes depend on
Ca2+ refilling into intracellular
Ca2+ stores, the depleted stores
are probably unable to keep Ca2+
oscillations. Prior addition of excess FKBP12 prevented the inhibitory effect of FK506. This suggests that extracellular FKBP12 may have trapped FK506 outside the cells, and hence FK506 could not penetrate into the cells and bind intracellular FKBP.
According to Cameron et al. (5), phosphorylation of
IP3R by protein kinase C increases
CRC activity in response to IP3, whereas dephosphorylation by calcineurin that is anchored to the IP3R via FKBP12 decreases CRC
activity. They suggested that reversible phosphorylation cycles in the
IP3R are important as a mechanism of ongoing Ca2+ oscillations. We
therefore assessed the effect of rapamycin and cyclosporin A to
elucidate whether the effect of FK506 is due to inhibition of
calcineurin. The addition of rapamycin inhibited Ca2+ oscillations as did FK506,
but cyclosporin A was without effect, suggesting that FK506 may exert
its effect by disturbing FKBP-IP3R association rather than by interfering with calcineurin-dependent dephosphorylation of IP3Rs. Thus
intracellular FKBP appears to be crucial for
Ca2+ oscillations.
Positive staining with anti-FKBP12 antibody confirmed the existence of
cytoplasmic FKBP in our cultured epithelium. However, it was difficult
to find a significant difference in immunocytochemical staining between
individual cells that showed oscillatory and nonoscillatory
Ca2+ responses induced by ATP or
between subconfluent and confluent cells. Thus a Western blotting
technique was employed to further investigate the difference in FKBP12
expression between our cultured epithelial cells at a subconfluent
stage, which tended to show long-lasting
Ca2+ oscillations, and those at a
confluent stage, which did not. We found that subconfluent cells had a
greater amount of FKBP12 than confluent cells as shown in Fig. 9.
Although this cannot readily explain the physiological relevance, we
speculate that FKBP12 may be one of the factors that regulates the
occurrence of Ca2+ oscillations.
In conclusion, FK506 is a potent immunosuppressant macrolide that
prevents graft rejection and autoimmune disorders, and this drug has
recently been shown to reduce allergic airway inflammation (16) and
airway hyperreactivity (9, 21). Our present data may add new
information about the pharmacological actions of FK506 that inhibit
Ca2+ oscillations, which may
result in the prevention of
Ca2+-mediated inflammatory
responses in airway epithelium.
 |
ACKNOWLEDGEMENTS |
We thank Yoshimi Sugimura and Masayuki Shino for technical
assistance and Fujisawa Pharmaceutical (Osaka, Japan) for providing FK506 and the anti-FKBP12 antibody (2C1).
 |
FOOTNOTES |
This work was supported in part by Grant 06670632 from the Ministry of
Education, Science and Culture, Japan.
Address for reprint requests and other correspondence: A. Nagai, First
Dept. of Medicine, Tokyo Women's Medical College 8-1 Kawada-cho,
Shinjuku-ku, Tokyo 162, Japan (E-mail:
a-nagai{at}tkd.att.ne.jp).
Received 23 October 1997; accepted in final form 11 February 1999.
 |
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