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Published ahead of print on August 28, 2003, doi:10.1165/rcmb.2003-0184OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 388-395, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0184OC

ATP Release Triggered by Activation of the Ca2+-Activated K+ Channel in Human Airway Calu-3 Cells

Yasushi Ito, Masami Son, Shinji Sato, Takayuki Ishikawa, Masashi Kondo, Shinsuke Nakayama, Kaoru Shimokata and Hiroaki Kume

Division of Respiratory Diseases, Department of Internal Medicine, and Department of Cellular Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan

Address correspondence to: Yasushi Ito, M.D., Division of Respiratory Diseases, Department of Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: itoyasu{at}med.nagoya-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Airway mucociliary clearance is subject to the autocrine/paracrine regulation of extracellular nucleotides released from the airway epithelial cells. The present study was performed in pursuit of effective modulators of ATP release under physiologic conditions in polarized human airway epithelial cells (Calu-3). Neither isoproterenol, forskolin, nor ionomycin augmented extracellular ATP release detected by luciferase assay. However, direct activation of the human intermediate conductance, Ca2+-activated K+ channel (hIK-1) by 1-ethyl-2-benzimdazolinone (1-EBIO, 1 mM) and chlorzoxazone (CZ, 1 mM) increased ATP release predominantly in the apical compartment. Measurement of fluo-3 signals revealed that 1-EBIO– and CZ-stimulated cytosolic Ca2+ mobilization was suppressed by the presence of MRS-2179, a specific P2Y1 receptor antagonist. The hIK-1–mediated ATP release was inhibited by a hIK-1 blocker (charybdotoxin), and an Na+-K+-2Cl- cotransport blocker (bumetanide) without interruption by GdCl3, an inhibitor of stretch-activated nonselective cation (SA) channels, or glybenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR). These results suggest that a cell volume decrease via the hIK-1–mediated KCl loss and the resultant induction of a regulatory volume increase via the Na+-K+-2Cl- transporter may trigger release of ATP, which causes P2Y1-mediated Ca2+ mobilization, through mechanisms unrelated to the CFTR and SA channels.

Abbreviations: 1-ethyl-2-benzimidazolinone, 1-EBIO • bumetanide, BMT • cystic fibrosis, CF • cystic fibrosis transmembrane conductance regulator, CFTR • charybdotoxin, ChTx • chlorzoxazone, CZ • chronic obstructive lung disease, COPD • 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, DIDS • forskolin, FK • human intermediate conductance, Ca2+-activated K+ channel, hIK-1 channel • short-circuit current, Isc • isoproterenol, ISO • intracellular Ca2+ concentration, [Ca2+]i • G protein–coupled purinergic receptors, P2Y • potential difference, PD • physiologic saline solution, PSS • regulatory volume increase, RVI • regulatory volume decrease, RVD • stretch-activated nonselective cation channels, SA channels • volume-sensitive large conductance ATP-permeable anion channel, VDACL


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is now well established that extracellular ATP and its metabolites induce a wide spectrum of biological responses through the activation of purinergic receptors via autocrine and paracrine mechanisms (1). On the airway surface, extracellular ATP released from airway epithelial cells regulates Cl- secretion, mucin secretion, and ciliary beat frequency to maintain a conductive and sterile environment in the respiratory tract (2–5). To date, several types of G protein–coupled purinergic receptors (P2Y), such as P2Y1, P2Y2, and P2Y4, have been detected in human airway epithelial cells (1). Thus, stimulation of P2Y on the airway surface has been recognized as a new approach to mucous congestive diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). From the clinical aspect, aerosolized purinergic receptor agonists are currently under development for this purpose (6). Alternatively, promotion of endogenous nucleotide release onto the airway surface also has potential as a therapy, although it has never been proposed. Thus, the present study was performed in pursuit of effective modulators of ATP release in polarized human airway epithelial cells.

In the cytosol, a significant amount (in the several millimolar range) of cytosolic ATP is in the form of Mg2ATP, and the free form of ATP (ATP4-) is in the micromolar range (7). Under physiologic conditions, there are at least three possible mechanisms for ATP release: the fusion of ATP-filled vesicles, conductive transport through ATP-permeable channels, and facilitated diffusion by ATP-specific anion transport (8). The effective triggers of ATP release are known to be mechanical stimuli, such as hypotonic shock, shear stress, and mechanical strain (8, 9). However, these stimuli are realistically difficult to apply as a clinical strategy for long-term management of the mucous congestive diseases. Recently, however, several laboratories have reported that ATP release was also stimulated by the cell volume decrease caused by hypertonic solutions (10, 11). Braunstein and coworkers (12) have demonstrated that dysfunction of a regulatory volume decrease, which occurs following a hypoosmolality-induced volume increase, impairs ATP release. It is well known that the process of a cell volume decrease involves outward K+ current flow followed by Cl- efflux, namely KCl loss (13). These findings suggest that activation of K+ channels might also trigger ATP release similar to hyperosmolality-induced mechanisms.

In excitable and nonexcitable cells, Ca2+-activated K+ (KCa) channels are ubiquitously expressed and regulate various physiologic responses by coupling cytosolic Ca2+ signaling (14). KCa channels have been classified as having large, small, or intermediate conductance based on their single channel conductance (14). Of these three types of KCa channels, intermediate conductance KCa channels are present in a variety of nonexcitable cells such as epithelial cells. RNA dot blot analysis has revealed a wide range in tissue expression of the channels in salivary gland, placenta, trachea, and lung (15). In various human epithelial cells such as airway and colon, the human intermediate conductance (10–31 pS), KCa (hIK-1) channel is abundantly expressed (16). Although hIK-1 channel openers, such as bendazolinones, are expected to provide a potential new therapy for the treatment of CF and COPD because of their strong Cl- secretory effects (17), the present study discovered novel effects of the hIK-1 channel openers on ATP release. The ATP release mechanisms seem likely to be different from those related to hypoosmotic stress.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
The Calu-3 cells (American Type Culture Collection, Rockville, MD) used in the present study are functionally and morphologically analogous to human airway serous cells expressing abundant CFTR on the apical membrane and hIK-1 on the basolateral membrane (16, 18, 19). These cells were grown in a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen), 100 µg/ml streptomycin, and 100 U/ml penicillin (Invitrogen) in culture flasks (T75) at 37°C in a humidified 95% air–5% CO2 atmosphere. When 80–90% confluent, cells were detached with a solution of phosphate-buffered saline (PBS), 0.04% EDTA, and 0.25% trypsin. The collected cells were passaged with a 1:4 dilution of the same solution and seeded onto porous polyester membranes (0.4 µm pore size on Snapwell or Transwell inserts [1.1 cm2; Costar, Cambridge, MA]) at a density of 106 cells/well. The inserts had been collagen-coated overnight with 0.2% human placental collagen type VI (Sigma-Aldrich, St. Louis, MO). The day after seeding the cells on the filters, the medium remaining on the apical side was removed to establish an air interface, which markedly improves the differentiation of the Calu-3 cells in a well-polarized fashion, forming the epithelial monolayer (20). The cells were fed by replacement of the basolateral medium every 48 h. Experiments were performed after 7–13 d in culture.

Solutions
The physiologic saline solution (PSS) used in the present studies was composed of (in mM): NaCl 115, KCl 5, MgCl2 1, CaCl2 2, glucose 10, Hepes 10, and NaHCO3 25. The pH of the solution was adjusted to 7.4 (at 37°C) by NaOH before addition of NaHCO3. The pH of the solution was kept at pH 7.4 when gassed with a mixture of 5% CO2, 21% O2, and 74% N2.

Luciferin/Luciferase ATP Assay
Unlike previous reports, we grew airway epithelial cells as polarized monolayers on permeable membrane supports in Transwell inserts (1 cm2) in twelve-well plates to measure ATP release from the apical and basolateral surfaces separately. This culture strategy yields an airway epithelial morphology that closely mimics in vivo morphology and permits access of experimental reagents to both epithelial aspects. Monolayers on the Transwell inserts were gently washed three times with PSS and were equilibrated for 30 min, maintaining the apical and basolateral solution volumes at 0.5 and 1 ml, respectively. The PSS was filtered with a Micropore membrane filter (0.22 µm) before experiments. ATP release was quantified by mixing 100 µl luciferase reagents with sample medium (100 µl) taken from apical and basolateral compartments after stimulation by various agents and their vehicle, using a luciferin/luciferase assay kit (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, a mixture of 100 µl sample and 100 µl luciferin/luciferase reagent solution was put into sample tubes, and the luminescent signal was measured with a Gene Light 55 luminometer (Yamato Scientific Co. Ltd., Tokyo, Japan). For maximum sensitivity, the light signal was integrated for 10 s after a delay of 1–2 s. Because of the mechanosensitivity of ATP release, sample collection was carefully and gently performed, and tilting culture plates was avoided (9). A calibration curve was constructed for each luciferase assay, using serial dilutions of an ATP standard. The calibration curve was plotted as the log of luminescence intensity (relative luminescence unit, RLU) versus the log of ATP concentrations (moles per liter). The ATP release was estimated by the amount of ATP from the 1 cm2 monolayer (pmol/cm2) at each time point (0, 2, 5 10, and 20 min after stimulation).

Measurement of [Ca2+]i
For measurement of [Ca2+]i, Calu-3 cells were subcultured on porous membranes of Transwell inserts (Costar) in 12-well plates. Before experiments, the apical and basolateral aspects of the confluent monolayer were rinsed twice with PSS and incubated for 1.5 h at 37°C in the same buffer containing 5 µM fluo-3/AM (Dojindo, Kumamoto, Japan) and 0.01% pluronic F127 (Molecular Probes, Eugene, OR). After the loading, cell monolayers were rinsed twice with PSS to wash off residual dyes outside the cells, and then 0.5 ml and 1 ml PSS were added to the apical and basolateral membranes, respectively. Fluorescence signals were collected for 20 ms at 6-s intervals using a fluorometer (Fluoroskan Ascent CF; Labsystems, Helsinki, Finland) at the excitation wavelength of 485 nm and the emission wavelength of 538 nm. The maximum signal (Fmax) was obtained by addition of 10 µM ionomycin, and the minimum signal (Fmin) was obtained by adding 10 mM EGTA to the cell monolayer. The [Ca2+]i was calculated according to the following formula:

in which Kd was assumed to be 390 nM.

Bioelectric Studies
The short-circuit current (Isc) measurements of cultured Calu-3 cells have been described previously (21). The Snapwell inserts where cells had grown confluent were rinsed with PSS and mounted in modified Ussing chambers (EasyMount Chamber; Physiologic Instruments, San Diego, CA) connected to a VCC MC2 voltage clamp (Physiologic Instruments). The chambers were filled with PSS and bubbled with 5% CO2, 21% O2, and 74% N2. The monolayers were continuously open-circuited to monitor transepithelial potential differences (PD), and every 20 s a bidirectional 2-µA pulse was imposed across the epithelium for 0.5 s to cause voltage deflections ({Delta} PD). This procedure allowed us to calculate transepithelial conductance (Gt) by Ohm's law (Gt= 2 µA/{Delta} PD). The short-circuit current (Isc) was recorded by clamping PD to 0 mV by VCC MC2. Isc represents the net flow of negative charges from the basolateral to the apical side. The Na+-K+-2Cl- cotransporter- and hIK-1–mediated ion currents were evaluated by measuring the Isc reduction in 10 min after application of bumetanide (BMT, 50 µM, basolateral) or charybdotoxin (ChTx, 100 nM, basolateral), respectively.

Reagents
Isoproterenol (ISO), ionomycin, forskolin (FK), chlorzoxazone (CZ), BMT, MRS-2179, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and glybenclamide were obtained from Sigma-Aldrich. 1-Ethyl-2-benzimidazolinone (1-EBIO) was purchased from Tocris Cookson Inc. (Ellisville, MO). ChTx and GdCl3 were products of Peptide Institute Inc. (Osaka, Japan) and Wako Chemical (Tokyo, Japan), respectively. ISO, GdCl3 and ChTx were dissolved in distilled water. All other drugs were dissolved in dimethylsulfoxide (DMSO). The final experimental medium after addition of these agents contained a maximum of 0.1% w/v DMSO.

Data Analysis
All data are expressed as means ± SEM, where n indicates the number of experiments. Statistical difference was determined by Student's t test or one-way ANOVA. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of Reagents on Luciferase Bioluminescence
Light output from the luciferase bioluminescence reaction is affected by a number of factors. For example, added agents may interfere with the reactions, hindering accurate ATP detection. Indeed, blockers of hypoosmolality-induced ATP release like DIDS and GdCl3 were reported to interfere directly with luciferase activity (8). To avoid misleading results, we ruled out possible interactions between luciferase reagents and drugs that were or would be applied to the apical solution in the present study. Thus, we measured the luciferase bioluminescence of the medium containing 0.1 nM ATP in the presence and absence of DMSO (0.1%), 1-EBIO (1 mM), CZ (1 mM), DIDS (100 µM), GdCl3 (30 and 500 µM), or glybenclamide (400 µM). As shown in Figure 1, DIDS, a putative blocker of swelling-activated ATP release, markedly interrupted the RLU obtained by 0.1 nM ATP (21.5 ± 2.2, n = 4, P < 0.001), compared with the control (318 ± 8.4, n = 6, Figure 1). GdCl3 at 30 µM, which blocks both stretch-activated nonselective cation channels (SA channels) and the volume-sensitive large conductance ATP-permeable anion channel (VDACL), did not affect the luminescence intensity (365 ± 34.7, n = 4), although a high concentration of this agent (500 µM) strongly prevented the luminescence (52.0 ± 8.9, n = 4, P < 0.001). The other drugs or vehicle DMSO (0.1%) had no significant effect on the luminescence.



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  		Figure 1. Direct effects of agents on luciferase bioluminescence. Luminescence intensity was expressed as relative luminescence units (RLU). The RLU was measured by gentle mixing of 100 µl luciferin/luciferase reagent and medium containing 0.1 nM ATP coexistent with DMSO (0.1%), 1-EBIO (1 mM), CZ (1 mM), DIDS (100 µM), GdCl3 (30 and 500 µM), and glybenclamide (400 µM) in sample tubes. DIDS (100 µM), known as a swelling-activated ATP-release blocker, markedly interrupted the RLU. GdCl3 (30 µM), which blocks both stretch-activated nonselective cation channels (SA channels) and the volume-sensitive large conductance ATP-permeable anion channel (VDACL), had no effect on the RLU in spite of luminescent interruption by a high concentration of this agent (500 µM). Data are means ± SEM (n = 4–6). Significant differences from the control values are expressed with *P < 0.001.

 
Effects of Anion Secretagogues on ATP Release
Under basal conditions, ATP released in the apical solution (0.68 ± 0.15 pmol/cm2, n = 29) was 10 times higher than that in the basolateral solution (0.07 ± 0.05 pmol/cm2, n = 28), suggesting that ATP is released rather predominantly on the apical membrane. To determine the effect of cAMP- and Ca2+-mediated anion secretagogues on ATP release, ATP released in apical and basolateral compartments was quantified 2, 5, 10, and 20 min after application of ISO (10 nM, basolateral), FK (10 µM, basolateral), or ionomycin (1 µM, basolateral). As shown in Figure 2, the application of ISO or FK did not cause a significant increase in the ATP release from the 1 cm2 monolayer in each compartment. Concretely, the average values of ATP released at 2, 5, 10, and 20 min after exposure to ISO and FK were 0.56 ± 0.08 (n = 20) and 0.43 ± 0.07 (n = 21) pmol/cm2 in the apical compartment and 0.078 ± 0.045 (n = 20) and 0.031 ± 0.008 pmol/cm2 (n = 20) in the basolateral compartment. On the other hand, the average ATP release in the presence of ionomycin (for 10 min) was rather less than that in its absence: 0.054 ± 0.009 (n = 15, P < 0.01) in the apical and 0.008 ± 0.002 pmol/cm2 (n = 13) in the basolateral compartment.



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  		Figure 2. Effects of anion stimulators on ATP release. The ordinate in A is expressed as a log scale, and that in B is a linear scale. Using luminometric assay, the time course of ATP release from 1 cm2 Calu-3 monolayer to apical (dotted bars) and basolateral (shaded bars) solutions was detected after exposure to isoproterenol (ISO, 10 nM, basolateral), forskolin (FK, 10 µM, basolateral), ionomycin (1 µM, basolateral), 1-EBIO (1 mM, bilateral), or CZ (1 mM, bilateral). The mixture of a 100-µl sample from each compartment and 100 µl luciferin/luciferase reagent was put in sample tubes, and the luminescent signal was measured with a luminometer at each time point to obtain the amount of released ATP using a calibration curve. Data are means ± SEM (n = 6–13).

 
Direct activators of hIK-1 channels such as 1-EBIO or CZ are also anion secretagogues (18, 22). To examine the possible involvement of the hIK-1 channel on the ATP release, the ATP assay was performed in the same way as described above after application of 1-EBIO (1 mM, bilateral) or CZ (1 mM, bilateral). As shown in Figure 2, increments in the released ATP in the extracellular solution were observed in the presence of the hIK-1 channel openers. The responses were found to start within a minute and to attain peaks at 10 min (70.7 ± 8.2 pmol/cm2, n = 13) with 1-EBIO and at 2 min (19.5 ± 8.0 pmol/cm2, n = 6) with CZ. The average values of ATP release at 2, 5, 10, and 20 min after exposure to 1-EBIO and CZ were 44.8 ± 7.1 and 12.7 ± 2.5 pmol/cm2 in the apical compartment and 13.7 ± 4.1 and 4.2 ± 0.8 pmol/cm2 in the basolateral compartment. These values were all significantly higher than the control at P < 0.001. Bilateral applications of DMSO (0.1%), which was the vehicle for 1-EBIO, CZ, and other agents, were without effect on the ATP release into the apical and basolateral solutions: 0.79 ± 0.14 (n = 8) and 0.01 ± 0.003 pmol/cm2 (n = 8) at 10 min after addition of this vehicle, respectively.

Ca2+ Signaling Dependent on the hIK-1 Channel
Reverse transcriptase–polymerase chain reaction studies to detect subtypes of P2Y receptors have revealed that only P2Y1 mRNA is expressed in Calu-3 cells (23). Next, we determined whether autocrine/paracrine stimulation via P2Y1 receptors was enhanced by the hIK-1 activation. As shown in Figure 3A, addition of 1-EBIO (1 mM) evoked intracellular Ca2+ oscillation, which was completely suppressed by pretreatment with MRS-2179 (100 µM, for 10 min in the bilateral solution), a specific P2Y1 receptor antagonist (Figure 3B). The behavior of [Ca2+]i in response to CZ was different from that to 1-EBIO. Sustained increases in [Ca2+]i were observed through the exposure to 1 mM CZ (Figure 3C). MRS-2179 also suppressed the [Ca2+]i increases caused by CZ, but infrequent [Ca2+]i responses remained (Figure 3D).



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  		Figure 3. Representative recordings of cytosolic Ca2+ concentrations ([Ca2+]i) estimated by fluo-3 signals. Application of 1-EBIO (1 mM, A) and CZ (1 mM, C) induced oscillatory increases in [Ca2+]i that were prevented by pretreatment with MRS-2179 (100 µM, for 10 min in the bilateral solution, B and D), a specific P2Y1 receptor antagonist.

 
Relationship between Bioelectric Properties and ATP Release
Applications of 1-EBIO (1 mM, bilateral) and CZ (1 mM, bilateral) led to a rapid increase in Isc followed by a sustained response (Figures 4A and 4B). The sustained components were reduced by addition of ChTx (100 nM, basolateral). The ChTx-sensitive Isc were increased by 1-EBIO and CZ (Figure 4C). The values under basal conditions (0.3 ± 0.1 µA/cm2, n = 4) were increased to 26.3 ± 5.2 µA/cm2 (n = 9, P < 0.01) and 11.5 ± 2.4 µA/cm2 (n = 11, P < 0.05) 20 min after addition of 1-EBIO and CZ, respectively. Also, the 1-EBIO– and CZ-induced responses were largely depressed by BMT (a Na+-K+-2Cl- cotransport inhibitor). The presence of these hIK-1 openers for 20 min augmented the BMT (50 µM)-sensitive components of Isc from 1.9 ± 0.4 µA/cm2 (n = 14) to 45.9 ± 9.8 (n = 6, P < 0.01) and 24.5 ± 8.2 µA/cm2 (n = 4, P < 0.01), respectively (Figure 4D). In contrast, ChTx caused a modest inhibition of the sustained increases in Isc in response to FK (Figure 5A). The ChTx-sensitive Isc estimated at 20 min after addition of ISO and FK were 1.3 ± 0.5 (n = 5) and 1.1 ± 0.4 µA/cm2 (n = 4), respectively, which were not significantly raised by these cAMP-related agents (Figure 5B). On the other hand, BMT-sensitive Isc (1.9 ± 0.4 µA/cm2, n = 14) was increased by ISO (6.9 ± 1.6 µA/cm2, n = 6, P < 0.01) and FK (8.1 ± 1.5 µA/cm2, n = 6, P < 0.01), as shown in Figure 5C. However, the BMT-sensitive components were minor in the total reactions. Although a Ca2+ ionophore, ionomycin, elicited hIK-1 channel-dependent increases in Isc, they were transient and immediately returned to the basal level (Figure 5D).



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  		Figure 4. Effects of hIK-1 openers on Isc. 1-EBIO (1 mM, A) and chlorzoxazone (CZ, 1 mM, B) stimulated sustained Isc that were not only sensitive to charybdotoxin (ChTx, 100 nM, basolateral, A, B, and C), but also to bumetanide (BMT, 50 µM, basolateral, D). Data are means ± SEM (n = 4–6). ChTx- and BMT-sensitive Isc were evaluated by measuring the Isc reduction in 10 min after the application of these inhibitors. Significantly different from the control with *P < 0.01 and **P < 0.05. #Indicates significant differences from the ChTx-sensitive or BMT-sensitive Isc produced by CZ by P < 0.05.

 


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  		Figure 5. Effects of cAMP- and Ca2+-related agents on Isc in Calu-3 cells. (A) Application of forskolin (FK, 10 µM, basolateral) biphasically increased Isc: a transient component followed by a sustained one that was steadily maintained for more than 20 min. ChTx (100 nM, basolateral) and bumetanide (BMT, 50 µM, basolateral) were added 20 and 30 min after starting exposure to FK to estimate ChTx- and BMT-sensitive Isc, respectively. ChTx- and BMT-sensitive Isc expressed the reduction in 10 min from the levels just before addition of these two inhibitors (B and C). With the same protocol, Isc in response to isoproterenol (ISO, 10 nM, basolateral) were followed (B and C). (D) A Ca2+ ionophore ionomycin (1 µM) elicited a transient increase in Isc that was immediately reduced to the basal level. The ionomycin-induced Isc was suppressed by pretreatment with ChTx. Data are means ± SEM (n = 4–6). Significantly different from the control with *P < 0.01.

 
Inhibition of hIK-1–Activated ATP Release
To clarify the involvement of hIK-1 and the Na+-K+-2Cl- cotransporter in the mechanisms underlying ATP release, ATP was assayed in the presence of ChTx or BMT. In the apical solution, the ATP released 10 min after addition of 1 mM 1-EBIO was 70.7 ± 8.2 pmol/cm2 (n = 4). The value was reduced to 30.5 ± 3.5 pmol/cm2 (n = 4, P < 0.05) or 12.3 ± 3.7 pmol/cm2 (n = 4, P < 0.01) by ChTx (100 nM, basolateral) or BMT (50 µM, basolateral) applied 10 min before 1-EBIO application (Figure 6). To further define the ATP release mechanisms in the hIK-1 activation, we examined the effects of agents that inhibit hypoosmolality-induced ATP release. However, GdCl3 (30 µM, apical) and glybenclamide (400 µM, bilateral) had no discernible effect on the extracellular ATP content (65.4 ± 12.3 pmol/cm2, n = 6 and 74.5 ± 16.9 pmol/cm2, n = 7, respectively, Figure 6).



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  		Figure 6. Effects of anion transport inhibitors on 1-EBIO-induced ATP release in the apical compartment of Calu-3 cells. Each column represents means ± SEM (n = 4–6). ATP content in the apical solution was measured 10 min after application of 1-EBIO (1 mM, bilateral) in the presence of ChTx (100 nM, basolateral), bumetanide (50 µM, basolateral), GdCl3 (30 µM, apical), or glybenclamide (400 µM, bilateral) for 30 min. Significant differences from the control values (only EBIO without any inhibitor) are expressed with *P < 0.05, **P < 0.01.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previously prevailing opinion was that the CFTR Cl- channel functions as a conductive pathway for ATP release, controlling the airway surface ATP concentration (24, 25). Nevertheless, patch-clamp studies of CFTR-expressing cells and planar bilayer studies of CFTR revealed no detectable ATP conductance (26, 27). The present study shows that activation of CFTR by isoproterenol or forskolin made no significant difference in ATP release in Calu-3 cells, in which abundant CFTR Cl- channels are expressed, indicating that CFTR actually failed to conduct ATP. Alternatively, the notion that the CFTR potentiates ATP release by a separate ATP channel under hypoosmotic conditions has been expressed (12). We demonstrated that these hIK-1 channel openers, 1-EBIO and CZ, are capable of facilitating ATP release, and that 1-EBIO–induced ATP release was unaffected by glybenclamide (400 µM), a selective CFTR blocker, which perfectly inhibits cAMP-induced Isc and ICFTR in Calu-3 cells (22, 28). These observations suggest the minor contribution of the CFTR to hIK-1–induced ATP release in this cell line.

Under the constitutive and hIK-1–stimulated conditions, intrinsic ATP appears to be released predominantly into the apical compartment, indicating that the ATP pathway seems likely to be present with an apically dominant distribution on the plasma membrane (see Figure 2). ATP released to the apical side stimulates apically localized P2Y receptors. It is well known that P2Y-mediated signaling pathway involves phospholipase C–mediated Ca2+ mobilization (1). Stimulation of P2Y receptors causes a [Ca2+]i increase in an oscillatory fashion in airway epithelial cells (29). In Calu-3 cells, which express only subtype P2Y1 (23), sustained anion secretion accompanied by intracellular Ca2+ oscillation is elicited especially by the apical application of our ATP and ADP (our unpublished data). In the present study, we applied a closed system rather than a perfusion system for [Ca2+]i measurements to detect the autocrine/paracrine mechanisms of extracellular ATP binding to the P2Y1 receptor. Specifically, in our system, extracellular solutions in the apical and basolateral compartments stayed put during an experiment, so that ATP released from the cells was able to stimulate the P2Y1 receptor. As expected, the hIK-1 activation by 1-EBIO induced cytosolic Ca2+ oscillation that was terminated by the selective P2Y1 receptor antagonist MRS-2179 (Figures 3A and 3B). On the other hand, CZ-induced Ca2+ responses, distinct from 1-EBIO–induced ones, were gradually incurred and sustained, but were similarly but incompletely inhibited by MRS-2179 (Figures 3C and 3D). This suggests that CZ may act not only as a hIK-1 activator but also as a Ca2+ mobilizer, although it has not been tested.

In most cells, regulation of cell volume is essential for survival under varying environmental and metabolic conditions (30). Hypoosmotic stress causes an acute cell volume increase and its subsequent gradual return to normal size, called the regulatory volume decrease (RVD) (31). The initial cell volume increase is generated by activation of Gd3+-sensitive stretch-activated nonselective cation channels (SA channels) (32). The subsequent RVD is ascribed to KCa channel-dependent KCl loss caused by Ca2+ entry through SA channels (13, 32). On the other hand, hyperosmotic conditions induce an acute cell volume decrease and its subsequent gradual return to normal size, called the regulatory volume increase (RVI) (31). Distinct from an initial volume increase in hypoosmolality, the solute influx in RVI is mediated either by the BMT-sensitive Na+-K+-2Cl- cotransporter or the coupled activities of the Na+-H+ exchanger and the Cl--HCO3- anion exchanger (32). Applications of hIK-1 openers, which initially cause a volume decrease by KCl loss, may mimic the hyperosmolality-induced cell responses. Indeed, 1-EBIO– and CZ-induced ChTx-sensitive Isc were abolished by the application of BMT (Figure 4), confirming that the BMT-sensitive Na+-K+-2Cl- cotransporter is greatly responsible for the RVI caused by hIK-1 openers. Recently, it has been reported that hypertonic as well as hypotonic solutions also facilitate ATP release in Jurkat T lymphocytes and Xenopus oocytes. (10, 11). The present study revealed that blocking the Na+-K+-2Cl- cotransporter reduces 1-EBIO–induced ATP release (Figure 6). Thus, the cotransporter-mediated RVI responses under the hIK-1-stimulated conditions may be involved in ATP release. In contrast, 1-EBIO–induced ATP release was unaffected by Gd3+ (30 µM), which is sufficient to inhibit SA channels (33, 34). This indicates little involvement of SA channels in the ATP release caused by hIK-1 activation.

In mouse mammary C127i cells, the hypoosmolality-induced cell volume increase activates volume-sensitive, large conductance, ATP-permeable anion channels (VDACL, ~ 400 pS) which are also blocked by 30 µM GdCl3 (35). However, Gd3+ (30 µM) had no effect on the luminescence of samples of the apical medium after stimulation by 1-EBIO (Figure 6). Therefore, it seems unlikely that VDACL is a conductive pathway for ATP under the hIK-1–activated condition.

Conventionally, luciferase assays have monitored extracellular ATP concentration in the bath rather than ATP efflux, and therefore the integrated response (i.e., ATP accumulation) has been reported. Recently, however, a surprising study using a medium perfusion system, which can monitor ATP efflux, revealed that hypoosmolality induces a biphasic ATP release composed of immediate and late reactions in Calu-3 cells (36). These results suggest that RVD is also involved in ATP release reactions, as reported by Braunstein and colleagues (12). In Jurkat T lymphocytes, a hypertonic solution causes rapid cell shrinkage, which is accompanied by cell deformation, leading to ATP release into the extracellular space (11). Simultaneous measurements of cell volumes and ATP release have revealed that a decrease in cell volume and ATP release are concurrently observed in hypertonic shock in Xenopus oocytes (10). Considering these data, hIK-1 channel-mediated cell shrinkage may also be involved in the ATP release from Calu-3 cells, although this was not tested in the present study.

In Calu-3 cells, most basolateral K+ conductance can be accounted for by the hIK-1 channel because only a small cAMP-activated K+ current, but a much larger hIK-1-dependent K+ current has been identified in the permeabilized Calu-3 monolayer (18, 37). Indeed, the findings in Figure 4 show that FK-, and ISO-induced Isc were insensitive to ChTx with a minor BMT-sensitive component. This may be one reason for the lack of effect on ATP release by these cAMP-related agents (see Figure 2).

The maximum concentrations of ATP were maintained for 20 min with difficulty even in the continual presence of the hIK-1 openers. This implies the involvement of surface-located ecto-nucleodases that rapidly degrade ATP to adenosine (38). That is, the amount of extracellular ATP would be reduced if the ATP release from the cells were subject to ATP degradation. Although the hIK-1 channel was also activated by ionomycin, the evoked anion current was transient (see Figure 5D), and extracellular ATP was rather decreased (see Figure 2). The inability to sustain the outward KCl current stimulated by ionomycin is probably due to massive Ca2+ influx that counteracts the driving force for Cl- export, leading to inhibition of KCl loss and following sustained RVI. That is, even if ATP were transiently released by ionomycin, the ATP degradation system would decrease extracellular ATP levels. Thus, it is more likely that continuous outward KCl current accompanied by NaCl and KCl uptake via BMT-sensitive cotransporter is necessary to maintain extracellular ATP levels.

Bodas and associates (39) have demonstrated that the ATP release estimated by luciferin/luciferase luminescence can be triggered in Xenopus oocytes by hyperpolarization pulses (from -60 mV to -200 mV) via nonexocytotic mechanisms. However, in Calu-3 cells, 1-EBIO (1 mM) causes only a small hyperpolarization (6 mV) of the apical membrane, and 16 mV hyperpolarization of the basolateral membrane (40) in spite of apical dominant ATP release (see Figure 2). Thus, it is less likely that hyperpolarization is involved in 1-EBIO–induced ATP release mechanisms.

Besides mechanosensitive ATP transport mechanisms, cell lysis is a possibility to increase extracellular ATP (8, 12). However, it seems unlikely because cell lysis was not evident when cells were examined microscopically and because BMT and ChTx inhibit ATP release stimulated by 1-EBIO.

In conclusion, the activation of the hIK-1 channel is a trigger for ATP release into the airway surface. Possibly, cell shrinkage via the hIK-1–stimulated KCl loss and RVI resulting from activation of the Na+-K+-2Cl- transporter may be implicated in this process. Treatment of respiratory complications of CF with gene therapy is not necessarily a feasible option because of the rapid turnover of epithelial cells of airways. Thus, pursuing pharmacologic therapy for pulmonary complications of CF and COPD may be more fruitful. Our results provide the clinically relevant information that the hIK-1 channel openers, as well as aerosolized P2Y receptor agonists, may relieve a variety of conditions in which mucociliary clearance is impaired, as seen in patients with CF and COPD.


   Acknowledgments
 
This work was supported by Research Grant Funds (#14770272) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to Y.I.

Received in original form May 7, 2003

Received in final form August 21, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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