Superoxide anion impairs Ca2+
mobilization in cultured human nasal epithelial cells
Tetsuya
Koyama1,2,
Masahiro
Oike1,
Sohtaro
Komiyama2, and
Yushi
Ito1
1 Department of Pharmacology
and 2 Department of
Otolaryngology, Faculty of Medicine, Kyushu University, Fukuoka
812-8582, Japan
 |
ABSTRACT |
We examined the effects of superoxide anion
(O
2) on the intracellular
Ca2+ concentration in cultured
human nasal epithelial cells. The cells were exposed to
O
2 by pretreatment with xanthine (X)
and xanthine oxidase (XO); control cells were treated with X alone.
When Ca2+-containing Krebs
solution was reperfused in the thapsigargin-treated, store-depleted
cells, reapplication-induced intracellular
Ca2+ concentration elevation was
significantly smaller in X/XO-treated cells than in the control cells,
suggesting that O
2 impairs
Ca2+ release-activated
Ca2+ entry (CRAC). Bath
application of ATP induced a steep
Ca2+ transient in both control and
X/XO-treated cells. However, the concentration-response curve of the
ATP-induced Ca2+ transient was
shifted to a higher concentration in X/XO-treated cells. The
impairments of CRAC and ATP-induced
Ca2+ transient induced by X/XO
were reversed by superoxide dismutase. Furthermore, all these
X/XO-induced effects were also observed in cells pretreated with
pyrogallol, also an O
2 donor. These
results indicate that O
2 impairs at
least two mechanisms involved in
Ca2+ mobilization in human nasal
epithelial cells, i.e., CRAC and ATP-induced
Ca2+ release.
capacitative calcium entry; adenosine 5'-triphosphate; calcium release
 |
INTRODUCTION |
NASAL EPITHELIUM REMOVES foreign particles from the
respiratory system by its ciliary movement and secretes mucin and
water. It has been reported that the ciliary beat frequency of airway epithelium is increased in proportion to the elevation in intracellular Ca2+ concentration
([Ca2+]i)
(13). The elevation in
[Ca2+]i
also results in the activation of the
Ca2+-dependent
Cl
channel, which
accompanies water secretion (7). Therefore, [Ca2+]i
has a significant role in controlling the functions of the nasal
epithelium. Voltage-dependent Ca2+
channels are not present in nonexcitable cells, including those in
airway epithelium, and mobilization of
Ca2+ is controlled mainly by
Ca2+ release from store sites and
the subsequent Ca2+
release-activated Ca2+ entry
(CRAC) (3, 10). There are a few reports on the
Ca2+ mobilization in human nasal
epithelial (HNE) cells. Lazarowski et al. (8) observed basically the
same Ca2+ phenomenon in HNE cells
as in other nonexcitable cells: i.e., purinergic agonist-induced
Ca2+ release via production of
inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3], thapsigargin-induced Ca2+ leak,
and CRAC. They found that HNE cells possess serosal P2U purinergic
receptors and mucinous P2U and unidentified UDP receptors, both of
which induce
[Ca2+]i
elevation. The same group also reported that
Ca2+ release and entry occurred
only at the same side of the cell to which the agonists were bound
(12). They speculated that this laterality of purinergic
receptor-mediated Ca2+
mobilization participates in the direction of
Cl
and water secretion.
Oxygen free radicals, which are generated at inflammatory sites (16),
have been reported to impair airway epithelial function. For example,
in tracheal organ culture, exposure to ozone increased the generation
of hydrogen peroxide
(H2O2),
which then increased the uptake of mineral particles by the tracheal
epithelium, which would damage the tissue (1). Furthermore, it was
reported that oxidant stress induced hypersecretion of mucin in
cultured guinea pig tracheal epithelium via a nitric oxide-dependent
mechanism (17). However, the details of these impairing effects of
reactive oxygen species have not been clarified. Because ciliary
movement as well as water secretion is controlled by
[Ca2+]i,
it could be speculated that Ca2+
homeostasis may be affected by reactive oxygen species.
Kimura et al. (4) have previously demonstrated the
impairing effects of superoxide anion
(O
2) on
[Ca2+]i
in vascular endothelial cells and found that
O
2 abolishes
Ca2+ oscillations by impairing
Ca2+ extrusion, CRAC, and a
Ca2+ leak pathway. It seems that
O
2 impairs endothelial function by
affecting Ca2+ mobilization
because the frequency of Ca2+
oscillations has been reported to be of critical importance in cellular
functions (9). However, there has been no study so far on the effect of
oxygen free radicals on
[Ca2+]i
in airway epithelium, especially nasal epithelium. Oxygen free radicals
including O
2 are generated by
neutrophils at inflammatory sites, and, therefore, it is of interest to
observe the action of O
2 on
[Ca2+]i
in HNE cells. We have therefore examined the effect of
O
2 on
Ca2+ mobilization in HNE cells,
and the results obtained suggest that alteration of
Ca2+ homeostasis may be involved
in reactive oxygen-induced damage of this tissue.
 |
METHODS |
Culture of HNE cells. HNE cells were
isolated from nasal polyps with the method described by Wu et al. (18).
Briefly, nasal polyps dissected by surgical operation were freed from
connective tissue and treated with 0.04% protease (Sigma, St. Louis,
MO) for 14 h at 4°C and thereafter with 0.1% trypsin (Sigma) for
10 min at 37°C. After gentle agitation, HNE cells were harvested by
centrifugation and seeded on Vitrogen (Nalgene, Palo Alto, CA)-coated
coverslips. Cells of either primary culture or one-step subculture were
then used for the experiments. We confirmed that there was no
difference in any of the Ca2+
responses between cells of primary culture and after one-step subculture.
Immunohistochemical staining of
keratin. We confirmed the epithelial nature of the
cells immunohistochemically with a method previously described (5).
Isolated and cultured cells prepared as described in
Culture of HNE cells
stained for keratin fibers (Fig. 1),
whereas fibroblasts from the connective tissue did not (data not
shown). Therefore, we regarded the cells obtained as above as HNE
cells, and these were used for the present experiments.

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Fig. 1.
Immunohistochemical staining of keratin fibers in human nasal
epithelial (HNE) cells. Cells were seeded either densely
(A) or sparsely
(B). Cytosolic keratin fibers were
stained in both conditions, suggesting epithelial nature of the
cells.
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Measurement of
[Ca2+]i
in HNE cells.
For the measurement of
[Ca2+]i
from a single isolated HNE cell (as shown in Fig.
1B), the cells grown on a coverslip
were loaded with 1 µM of the acetoxymethyl ester form of
the Ca2+ fluorescent dye fura 2 (fura 2-AM; Wako, Osaka, Japan) for 20 min at room temperature and
thereafter for 20 min at 37°C. The coverslip with fura 2-loaded
cells was then placed in a chamber of 0.5-ml volume and mounted on an
inverted microscope (Diaphot TMD, Nikon, Tokyo, Japan). The cell was
excited with two excitation wavelengths, 340 and 380 nm (each slit 5 nm) applied by a spectrometer (Spex, Edison, NJ). The obtained
fluorescent intensity data after subtraction of the background
fluorescence were used to obtain the fluorescence ratio (R = fluorescence at 340 nm to that at 380 nm). We calculated the apparent
[Ca2+]i
using the equation
[Ca2+]i = Keff[(R
Rmin)/(Rmax
R)], where
Keff is the
effective binding constant, Rmin
is the R at 0 Ca2+ and
Rmax is the R at high
Ca2+. Because precise in vivo
calibration of
[Ca2+]i
was difficult to perform, it should be noted that the calculated value
is not an accurate
[Ca2+]i value.
In some experiments, we calculated the net
Ca2+ mobilized by integrating the
elevated component of
[Ca2+]i
(
[Ca2+]i),
i.e., the time integral of
[Ca2+]i
(
[Ca2+]i · dt).
In other experiments, we estimated the maximum rate of
Ca2+ leak induced by thapsigargin
by differentiating
[Ca2+]i,
i.e.,
d[Ca2+]i/dt.
Both calculations were performed with Microsoft Excel.
Materials. Modified Krebs solution
containing (in mM) 132 NaCl, 5.9 KCl, 1.5 CaCl2, 1.2 MgCl2, 11.5 glucose, and 11.5 HEPES was used as the standard extracellular
solution; pH was adjusted to 7.3 with NaOH.
Ca2+-free solution was made by
substituting CaCl2 with 1 mM EGTA. The bath was continuously perfused with the solution at a rate of 1.5 ml/min. All experiments were performed at room temperature.
ATP and thapsigargin were used to release
Ca2+ from the intracellular
Ca2+ store sites. Stock solutions
of xanthine (X), xanthine oxidase (XO), superoxide dismutase (SOD), and
pyrogallol were diluted 1,000 times to make the final
concentrations. All these drugs were obtained from Sigma (St.
Louis, MO).
Data analysis. Pooled data are given
as means ± SE, and significance was determined with Student's
unpaired t-test. P < 0.05 was regarded as significant.
 |
RESULTS |
Effects of O
2 on basal level
of
[Ca2+]i
in HNE cells.
First, we examined the effect of O
2 on
the basal level of
[Ca2+]i
in HNE cells that were not stimulated by any
Ca2+-mobilizing agents.
O
2 was applied to the cells by bath
perfusion of normal Krebs solution containing 200 µM X and 20 mU/ml
of XO or by pretreatment of the cells with X/XO. As shown in Fig.
2A, bath
application of X/XO for up to 20 min did not induce any change in
[Ca2+]i.
The resting
[Ca2+]i
after incubation of HNE cells with X/XO for 45 min at 37°C was also
not significantly different from that in control cells (Fig.
2B). Thus it appears that
O
2 does not influence the resting
level of
[Ca2+]i
in HNE cells, at least up to 45 min. Furthermore, this indicates that
continuous bath perfusion itself, which would generate some mechanical
stress such as shear stress, does not induce any change in
[Ca2+]i
in the present experiment.

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Fig. 2.
Resting level of intracellular
Ca2+ concentration
([Ca2+]i)
in HNE cells and effect of xanthine/xanthine oxidase (X/XO).
A: bath application of 200 µM X and
20 mU/ml XO did not induce any change in
[Ca2+]i.
B: cells were incubated with Krebs
solution alone (untreated), 200 µM X alone, or 200 µM X and 20 mU/ml XO for 45 min at 37°C. Then
[Ca2+]i
was measured. There was no significant difference between any of these
values. Nos. in parentheses, no. of cells.
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Effect of O
2 on
Ca2+ leak and
CRAC.
We then examined the effect of O
2 on
thapsigargin-induced Ca2+
mobilization. Thapsigargin, an inhibitor of endoplasmic
Ca2+-ATPase, is known to deplete the intracellular
Ca2+ store sites by Ca2+ leak (14). In control
cells (pretreated with X alone), bath application of 1 µM
thapsigargin in a Ca2+-free solution induced a transient
elevation in [Ca2+]i. When a
Ca2+-containing solution was applied to these
store-depleted cells, a gradual elevation in
[Ca2+]i was elicited (Fig.
3A). On the other hand, when
thapsigargin was applied to X/XO-treated cells, an initial elevation in
[Ca2+]i was observed as in control
cells, whereas the following application of extracellular
Ca2+ failed to induce a further
[Ca2+]i elevation (Fig.
3B).

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Fig. 3.
Thapsigargin-induced
[Ca2+]i
elevation in HNE cells. Thapsigargin (1 µM) was applied (arrows) in
Ca2+-free solution to cells
treated with X alone (A) or after
pretreatment with X/XO for 45 min at 37°C
(B). Subsequent application of
extracellular Ca2+ induced
[Ca2+]i
elevation in A but no
[Ca2+]i
elevation in B.
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We then analyzed the thapsigargin-induced
Ca2+ response. Differentiation of
the initial Ca2+ transient
(d[Ca2+]i/dt),
which yields the velocity of Ca2+
leak, showed maximum values of 1.02 ± 0.14 and 1.51 ± 0.20 nM/s in X/XO-treated (n = 8)
and control (n = 12) cells,
respectively (P > 0.05).
Furthermore,

[Ca2+]i · dt
of the initial Ca2+ transient was
also not significantly different between the cells treated with X/XO
and X alone (P > 0.05; Fig.
4B). On the other hand, a
Ca2+ reapplication-induced peak
[Ca2+]i
elevation after store depletion
(
[Ca2+]i,CRAC)
was significantly smaller in X/XO-treated cells than in control cells
(X/XO: 7.8 ± 3.9 nM, n = 10 cells;
X alone: 60.3 ± 17.0 nM, n = 12 cells; P < 0.01; Fig.
4C). This indicates that CRAC was
significantly affected by treatment with X/XO.
To confirm that the effects of X/XO were not due to its nonspecific
action, we examined the effect of SOD and pyrogallol on thapsigargin-induced Ca2+
mobilization. If 150 U/ml of SOD are present during the incubation period with X/XO, O
2 is expected to be
scavenged. In such a condition,

[Ca2+]i · dt
was also not significantly different from control or X/XO-treated cells
(Fig.
4B),
but
[Ca2+]i,CRAC
was reversed (54.1 ± 13.4 nM; P > 0.05 compared with X alone; n = 6 cells;
Fig. 4C). When the cells were
incubated with 10 µM pyrogallol, another
O
2 generator, for 45 min at 37°C,
bath application of thapsigargin induced similar changes in
Ca2+ mobilization.

[Ca2+]i · dt
of the thapsigargin-induced initial
Ca2+ peak was not affected (Fig.
4B), but
[Ca2+]i,CRAC
was significantly impaired by pyrogallol (16.8 ± 3.3 nM; P < 0.05 compared with X alone;
n = 10 cells; Fig.
4C).

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Fig. 4.
Analysis of thapsigargin-induced
[Ca2+]i
elevation. A: measured parameters:
time integral of elevated
[Ca2+]i
( [Ca2+]i · dt)
of thapsigargin-induced Ca2+ leak
in Ca2+-free solution and peak
[Ca2+]i
elevation induced by Ca2+
reapplication
( [Ca2+]i,CRAC),
where CRAC is Ca2+
release-activated Ca2+ entry.
There was no significant difference in
 [Ca2+]i · dt
among groups (B). SOD, superoxide
dismutase.
[Ca2+]i,CRAC
was significantly decreased in X/XO- and pyrogallol (10 µM)-treated
cells compared with that in X alone-treated cells
(* P < 0.05;
** P < 0.01), whereas in
X/XO+SOD (150 U/ml)-treated cells, it was not significantly different
from that in X alone-treated cells
(C). Nos. in parentheses, no. of
cells.
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Thus it seems plausible to assume that the effects of X/XO on
thapsigargin-induced Ca2+
mobilization were due to the generation of
O
2 but not to its nonspecific action
by other catalytic products of the X/XO reaction.
Effect of O
2 on ATP-induced
[Ca2+]i
elevation.
HNE cells have been reported to possess
P2U receptors and
produce the
[Ca2+]i
response by the production of
Ins(1,4,5)P3 (8),
and ATP is an important chemical mediator of many biological reactions. We therefore examined the effect of ATP on
[Ca2+]i
in HNE cells. In control cells treated with X alone for 45 min at
37°C, bath application of ATP induced an
[Ca2+]i
elevation, with a threshold of 0.1 µM in a
Ca2+-containing solution (Fig.
5Aa).
ATP-induced
[Ca2+]i
elevation was always transient, and
Ca2+ oscillations were rarely
observed with any concentration of ATP (Fig.
5A). For the quantitative
analysis of the ATP-induced
Ca2+ transient, we used
two factors: peak
[Ca2+]i
elevation
(
[Ca2+]i,peak)
and

[Ca2+]i · dt.
[Ca2+]i,peak,
which would correlate mainly to ATP-induced
Ca2+ release, increased in a
concentration-dependent manner, with an
EC50 value of 4.6 ± 1.1 µM
and a maximal
[Ca2+]i,peak
of 212.8 ± 12.1 nM (Fig.
6A).

[Ca2+]i · dt,
which would reflect overall Ca2+
mobilization by ATP, also showed a concentration dependency, with an EC50 of 3.9 ± 0.9 µM and a maximal

[Ca2+]i · dt
of 42.8 ± 2.9 µM · s (Fig.
6B).

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Fig. 5.
ATP-induced
[Ca2+]i
elevation in HNE cells. A: indicated
concentrations of ATP were applied to cells treated with X alone in
Ca2+-containing Krebs solution.
Note that a slight
[Ca2+]i
increase was elicited by as low as 0.1 µM ATP, and
Ca2+ oscillations were not induced
by any of these concentrations. B: ATP
was applied to X/XO-treated cells. Note that 0.3 µM ATP did not
induce
[Ca2+]i
elevation.
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Fig. 6.
Concentration-response relationship of ATP-induced
Ca2+ transient.
A: peak
[Ca2+]i
elevation
( [Ca2+]i,peak)
induced by ATP. Continuous and dotted lines were drawn by a Boltzmann
equation with half-maximal ATP concentrations ([ATP]) of
4.6 and 16.5 µM and maximum
[Ca2+]i,peak
values of 212.8 and 205.2 nM for control ( ) and X/XO-treated ( )
cells, respectively. B:
 [Ca2+]i · dt
of elevated ATP-induced Ca2+
transient for 5 min. Continuous and dotted lines were drawn with
half-maximal ATP concentrations of 3.9 and 19.0 µM and maximum
 [Ca2+]i · dt
values of 42.8 and 38.5 µM · s for control
(X-alone; ) and X/XO-treated ( ) cells, respectively. ,
X/XO+SOD-treated cells; , pyrogallol-treated cells; ,
pyrogallol+SOD-treated cells.
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When cells were pretreated with X/XO for 45 min at 37°C, bath
application of ATP also induced a
[Ca2+]i
elevation, but its threshold was higher than that in control cells. A
low concentration of ATP (0.3 µM) did not evoke a
Ca2+ transient (compare Fig.
5Ba with Fig.
5Ab). The pattern of
Ca2+ transient induced by a higher
concentration of ATP was similar to that in control cells; i.e., no
Ca2+ oscillations were observed
(Fig. 5Bb). The
concentration-response relationships of the ATP-induced
Ca2+ transient were shifted to the
right, with EC50 values of 16.5 ± 6.4 and 19.0 ± 4.2 µM for
[Ca2+]i,peak
and

[Ca2+]i · dt,
respectively (Fig. 6). The maximal
[Ca2+]i,peak
was similar to the control value (205.2 ± 20.6 nM), and the maximal

[Ca2+]i · dt
was slightly smaller than the control value (38.5 ± 2.8 µM · s).
It is possible that the impaired

[Ca2+]i · dt
may be due to the alteration of
Ca2+ extrusion; i.e.,

[Ca2+]i · dt
would be increased and decreased when
Ca2+ extrusion is suppressed and
accelerated, respectively. So we then applied 100 µM ATP in a
Ca2+-free solution and calculated
the time required to decrease
[Ca2+]i
to one-third of the peak
(t1/3) as an
indicator of Ca2+ extrusion.
ATP-induced Ca2+ transient showed
t1/3 values of
155.3 ± 12.7 s in control cells (n = 8) and 145.6 ± 13.4 s in X/XO-treated cells
(n = 7;
P > 0.05). Therefore, it seems that
the Ca2+ extrusion mechanism is
not affected by treatment with X/XO and that the rightward shift of the

[Ca2+]i · dt
curve can be attributed to impairment of
[Ca2+]i,peak
and CRAC.
We then examined the effects of SOD on this impairing action of X/XO.
When cells were pretreated with X/XO in the presence of 150 U/ml of
SOD, both

[Ca2+]i · dt
and
[Ca2+]i,peak
values of the 10 µM ATP-induced
Ca2+ transient were not
significantly different from those of control cells (X alone; Fig. 6,
Table 1). On the other hand, pyrogallol induced similar alterations to ATP-induced
Ca2+ mobilization as in
X/XO-treated cells, and

[Ca2+]i · dt
and
[Ca2+]i,peak
were significantly smaller than in the control cells. The effects of
pyrogallol on
[Ca2+]i,peak
and

[Ca2+]i · dt
were also reversed by SOD (Fig. 6, Table 1).
These results indicate that the effects of X/XO on ATP-induced
Ca2+ transient were due to the
production of O
2.
 |
DISCUSSION |
In this study, we examined the effects of
O
2 on
Ca2+ homeostasis in HNE cells.
When the cells were pretreated with X/XO, CRAC and ATP-induced
Ca2+ release were impaired. These
effects of X/XO on Ca2+
mobilization were not nonspecific actions but were produced by the
production of O
2. This view is
supported by the following observations:
1) SOD reversed the impairing
effects of X/XO; 2) pyrogallol,
another O
2 donor, mimicked all of the
impairing effects of X/XO; and 3)
the effects of pyrogallol were also reversed by SOD.
In the previous report on aortic endothelial cells, Kimura et al. (4)
showed that O
2 inhibits CRAC and the
plasmalemmal Ca2+ pump and
accelerates Ca2+ leak from
intracellular store sites, thereby inhibiting
Ca2+ oscillations. In HNE cells,
however, the Ca2+ leak pathway was
not affected by O
2 because the maximum
leak velocity
(d[Ca2+]i/dtmax
of thapsigargin-induced
[Ca2+]i
elevation) was not significantly different between control and
X/XO-treated cells. Furthermore, the
Ca2+ extrusion mechanism was not
impaired by O
2 either because the
t1/3 of the
ATP-induced Ca2+ transient in a
Ca2+-free solution was not
significantly different between control and X/XO-treated cells.
Therefore, it is reasonable to assume that the only common change
induced by O
2 in HNE and bovine aortic
endothelial cells is the impairment of CRAC (Fig.
4C), which is the most important
Ca2+ entry pathway in HNE cells
(8).
Because Ca2+ extrusion and
Ca2+ leak velocity were not
significantly affected by treatment with X/XO,

[Ca2+]i · dt
of the thapsigargin-induced initial
Ca2+ transient would correlate to
the total amount of stored Ca2+.
This value was also not altered by O
2
(Fig. 4B), thereby indicating that
O
2 does not change the total amount of
stored Ca2+. Furthermore, the
resting
[Ca2+]i
was not altered by X/XO either (Fig. 2). So it seems that
O
2 mainly affects the stimulated
Ca2+ mobilizations in HNE cells,
at least up to 45 min.
Another impairing effect of O
2 on
Ca2+ mobilization in HNE cells was
the reduction in ATP-induced
[Ca2+]i,peak
(Figs. 5 and 6A). Because the
reduction in
[Ca2+]i,peak
was observed in both
Ca2+-containing (Fig. 5) and
Ca2+-free (Table 1) solutions, it
seems that ATP-induced Ca2+
release was impaired by O
2. This is
also supported by the fact that the threshold of the ATP-induced
Ca2+ transient was shifted to a
higher concentration by X/XO (Figs. 5Ba and
6A). But probably because the total
amount of stored Ca2+ was not
changed by O
2, the maximal
[Ca2+]i,peak
was similar to the control value (Fig.
6A). In this study, we could not
clarify the mechanism by which O
2 impaired ATP-induced Ca2+ release.
One possible mechanism is the inhibition of
Ins(1,4,5)P3 production, and another is the impairment of the
Ca2+ release pathway. In coronary
artery smooth muscle cells, Ca2+
release induced by exogenously applied
Ins(1,4,5)P3 was
inhibited by O
2 (2). On the other
hand, norepinephrine-induced contraction in the rabbit mesenteric
artery was impaired by oxygen free radicals by inhibiting
Ins(1,4,5)P3
production (15). Therefore, impairment of both
Ins(1,4,5)P3
production and Ca2+ release by
Ins(1,4,5)P3 may
be responsible for the attenuation of ATP-induced
Ca2+ release by
O
2 in HNE cells. Further investigation is needed to clarify the detailed mechanism of the impairing action of
O
2 on ATP-induced
Ca2+ release.
It can be speculated that both impairments of
Ca2+ mobilization, i.e.,
ATP-induced Ca2+ release and the
subsequent CRAC, were responsible for the reduction in ATP-induced

[Ca2+]i · dt
by O
2 (Figs. 5 and
6B). In general, there are at least
two modulation patterns of
[Ca2+]i
in response to agonists in nonexcitable cells: i.e.,
Ca2+ oscillations (4) and
sustained
[Ca2+]i
elevation (11). HNE cells did not show
Ca2+ oscillations at any
concentration of ATP, but
[Ca2+]i,peak
and

[Ca2+]i · dt
depended on ATP concentration (Figs. 5 and 6). The reason why HNE cells
did not show Ca2+ oscillations is
unclear in the present experiments. However, such an "amplitude
modulation" of
[Ca2+]i
may have a functional significance in HNE cells because it has been
reported that the ciliary beat frequency of rabbit airway epithelium
depends on the sustained level of
[Ca2+]i
(6). Therefore, the impairments of CRAC and
Ca2+ release by
O
2 and the resultant reduction in agonist-induced

[Ca2+]i · dt
would cause severe damage to the cellular functions of HNE cells.
Oxygen free radicals such as O
2 and
H2O2
are generated by polynucleated neutrophils at inflammatory sites (16).
H2O2
causes the same impairing effect on
Ca2+ mobilization as
O
2 in HNE cells (Koyama and Oike,
unpublished observation). HNE cells protect airways from environmental
particles such as dusts or bacteria. Therefore, the impairment of
Ca2+ homeostasis by oxygen free
radicals shown in the present study would be related to the
pathogenesis of inflammatory disease of the upper airway such as
sinusitis. Because SOD restored all of the
O
2-induced impairment of
Ca2+ mobilization almost
completely, scavenging oxygen free radicals might become a novel
approach for the treatment of inflammatory disease of the upper airways.
In summary, we have investigated the effect of
O
2 on
Ca2+ homeostasis in HNE cells and
found that CRAC pathway and ATP-induced Ca2+ release are affected.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Kate E. Creed for critical reading of the manuscript.
 |
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: M. Oike, Dept.
of Pharmacology, Kyushu Univ., Fukuoka 812-8582, Japan (E-mail:
moike{at}pharmaco.med.kyushu-u.ac.jp).
Received 4 May 1999; accepted in final form 6 August 1999.
 |
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