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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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 (Delta [Ca2+]i), i.e., the time integral of Delta [Ca2+]i (<LIM><OP>∫</OP></LIM>Delta [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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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, <LIM><OP>∫</OP></LIM>Delta [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 (Delta [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, <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt was also not significantly different from control or X/XO-treated cells (Fig. 4B), but Delta [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. <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt of the thapsigargin-induced initial Ca2+ peak was not affected (Fig. 4B), but Delta [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 (<LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt) of thapsigargin-induced Ca2+ leak in Ca2+-free solution and peak [Ca2+]i elevation induced by Ca2+ reapplication (Delta [Ca2+]i,CRAC), where CRAC is Ca2+ release-activated Ca2+ entry. There was no significant difference in <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt among groups (B). SOD, superoxide dismutase. Delta [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.

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 (Delta [Ca2+]i,peak) and <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt. Delta [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 Delta [Ca2+]i,peak of 212.8 ± 12.1 nM (Fig. 6A). <LIM><OP>∫</OP></LIM>Delta [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 <LIM><OP>∫</OP></LIM>Delta [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 (Delta [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 Delta [Ca2+]i,peak values of 212.8 and 205.2 nM for control (open circle ) and X/XO-treated () cells, respectively. B: <LIM><OP>∫</OP></LIM>Delta [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 <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt values of 42.8 and 38.5 µM · s for control (X-alone; open circle ) and X/XO-treated () cells, respectively. , X/XO+SOD-treated cells; black-triangle, pyrogallol-treated cells; triangle , pyrogallol+SOD-treated cells.

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 Delta [Ca2+]i,peak and <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt, respectively (Fig. 6). The maximal Delta [Ca2+]i,peak was similar to the control value (205.2 ± 20.6 nM), and the maximal <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt was slightly smaller than the control value (38.5 ± 2.8 µM · s).

It is possible that the impaired <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt may be due to the alteration of Ca2+ extrusion; i.e., <LIM><OP>∫</OP></LIM>Delta [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 <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt curve can be attributed to impairment of Delta [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 <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt and Delta [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 <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt and Delta [Ca2+]i,peak were significantly smaller than in the control cells. The effects of pyrogallol on Delta [Ca2+]i,peak and <LIM><OP>∫</OP></LIM>Delta [Ca2+]i · dt were also reversed by SOD (Fig. 6, Table 1).

                              
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Table 1.   Maximal Delta  [Ca2+]i and int Delta [Ca2+]i · dt values of ATP-induced Ca2+ transient under various conditions

These results indicate that the effects of X/XO on ATP-induced Ca2+ transient were due to the production of O-2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, <LIM><OP>∫</OP></LIM>Delta [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 Delta [Ca2+]i,peak (Figs. 5 and 6A). Because the reduction in Delta [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 Delta [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 <LIM><OP>∫</OP></LIM>Delta [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 Delta [Ca2+]i,peak and <LIM><OP>∫</OP></LIM>Delta [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 <LIM><OP>∫</OP></LIM>Delta [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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(6):L1089-L1095
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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