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Published ahead of print on September 11, 2003, doi:10.1165/rcmb.2003-0183OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 411-419, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0183OC

Apical and Basolateral ATP-Induced Anion Secretion in Polarized Human Airway Epithelia

Masami Son, Yasushi Ito, 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
 
The present study investigated mechanisms underlying apical and basolateral P2Y1-mediated Cl- secretion in human airway epithelial cells. Apical and basolateral ATP induced short-circuit currents (Isc) with different properties via P2Y1 receptors. The former comprised an immediate rise followed by a slow attenuation, whereas the latter was a transient rise with a higher peak and shorter duration (< 2 min). The actions of ATP were simulated by those of ADP, ADPßS, and ATP{gamma}S. Antagonists of phosphatidylinositol-phospholipase C (U73122, ET-18-OCH3) were without any effect on the bilateral ATP-induced Isc, which were, in contrast, attenuated by a phosphatidylcholine-phospholipase C inhibitor (D609) and an adenylate cyclase inhibitor (SQ22536). The responses to ATP from either aspect were also sensitive to an intracellular Ca2+ chelator, 1,2-bis (o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester, or a Ca2+-activated K+ channel inhibitor, charybdotoxin, although differential Ca2+ signals were concomitant with each reaction. Nystatin permeabilization studies revealed a good correlation between the Isc and the basolateral K+ current rather than the apical Cl- current under ATP-stimulated conditions. In conclusion, apical and basolateral P2Y1 receptors couple with both phosphatidylcholine-phospholipase C and adenylate cyclase, leading to Cl- secretion, whose rate is essentially regulated by the Ca2+-activated K+ channel–mediated K+ conductance. This suggests the importance of this channel in airway mucociliary clearance.

Abbreviations: adenylate cyclase, AC • 1,2-bis (o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester, BAPTA-AM • intracellular Ca2+ concentration, [Ca2+]i • cystic fibrosis transmembrane conductance regulator, CFTR • charybdotoxin, ChTx • diacylglycerols, DAG • human intermediate conductance, inward-rectifying Ca2+-activated K+ channel, hIK-1 channel • apical Cl- current, ICl • basolateral K+ current, IK • short-circuit current, Isc • Ca2+-activated K+ channel, KCa channel • 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB • phosphate-buffered saline, PBS • phosphatidylcholine-phospholipase C, PC-PLC • potential difference, PD • phosphatidylinositol-phospholipase C, PI-PLC • protein kinase C, PKC • physiologic saline solution, PSS • transepithelial resistance, Rt • uridine 5'-triphosphate, UTP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The electrolyte transport system, Na+ absorption and Cl- secretion, across the airway epithelia followed by water movement through the paracellular pathway is essential for the normal mucociliary clearance that maintains the aseptic condition of the respiratory tract (1). Recently, a great deal of attention has been paid to the physiologic role of extracellular ATP and other nucleotides in a wide spectrum of biological responses, including the electrolyte transport through specific receptors called "purinoreceptors" on the outside of the plasma membrane (2). Pharmacologic and molecular biology studies have identified two types of purinoreceptors: P1 and P2. P1 receptors (adenosine receptors) are further subdivided into four types: A1, A2A, A2B, and A3. On the other hand, P2 receptors (ATP receptors) are subclassified as ionotropic P2X receptors and metabotropic P2Y receptors (2). To date, seven mammalian P2X receptors (P2X1–7), which are ligand-gated monovalent cation channels, and five mammmalian P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11), which are G protein–coupled receptors, have been cloned (2). Among these receptors, P2Y1, P2Y2, P2Y4, and P2Y6 have been detected in human airway epithelial cells (3, 4). The major roles of extracellular nucleotides bound to the airway P2Y receptors are upregulation of Cl- and mucin secretion as well as ciliary motility (5–7). Based on this fact, aerosolized nucleotides have been proposed as a potential therapy for mucous congestive diseases such as cystic fibrosis (CF) and chronic obstructive lung disease (8).

In polarized nasal and pancreatic epithelial cells, nucleotide receptors are located on both apical and basolateral membranes (4, 9). However, notwithstanding much endeavor to clarify the roles of luminal P2Y receptors on airway epithelial cells, little is known regarding the effects of the basolateral P2Y receptors. A recent study has reported that apical and basolateral P2Y (P2Y1 and P2Y2) receptors cooperatively regulate Ca2+ mobilization in nasal epithelial cells (4). In pancreatic duct cells, it has been shown that apical and basolateral P2Y receptors have opposite effects on fluid secretion (9). Thus, the present study was designed to compare the effects of apical and basolateral P2Y-mediated signal transduction pathways linking to transepithelial anion transport, using polarized Calu-3 cells which may be a model of human airway submucosal gland serous cells (10). This cell line expresses abundant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels on the apical membrane and human intermediate conductance (10–31 pS), inward-rectifying Ca2+-activated K+ (hIK-1) channels on the basolateral membrane (11–13). Here we demonstrate pharmacologic evidence that application of apical and basolateral ATP induced different types of anion secretion resulting from the distinct behaviors of the hIK-1 channel on the basolateral membrane.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Calu-3 human airway cells (American Type Culture Collection, Manassas, VA) at passages 29–35 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). The cells were incubated in culture flasks (T75) at 37°C in a humidified atmosphere of 5% CO2 in air. 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 inserts [1.1 cm2]; Costar, Cambridge, MA) at a density of 106 cells/well. The Snapwell 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 human airway epithelia in a well-polarized fashion (14). 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) contained (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) using NaOH before addition of NaHCO3. During the experiments, the solution was gassed with a mixture of 5% CO2, 21% O2, and 74% N2 to keep the pH at pH 7.4. This solution was bubbled with 5% CO2, 21% O2, and 74% N2.

Bioelectric Studies
When cells had grown confluent, the Snapwell inserts 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 monolayers were continuously open-circuited to monitor transepithelial potential differences (PD), and every 5–20 s a bidirectional 2-µA pulse was imposed across the epithelium for 0.5 s to cause voltage deflections ({Delta} PD). This procedure enabled us to calculate transepithelial resistance (Rt) by Ohm's law (Rt = {Delta} PD/2 µA). 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.

Measurement of Apical Cl- Current and Basolateral K+ Current
To assess Cl- current across the apical membrane (ICl), an apical-to-basolateral Cl- concentration gradient was established by replacing NaCl with equimolar Na-gluconate in the basolateral PSS. For this measurement, the basolateral membrane was permeabilized with the pore-forming antibiotic nystatin (100 µM) for more than 30 min. This procedure avoids the complexities associated with basolateral ion transporters and permits analyses of apical membrane Cl- conductance (12, 15). Complete permeabilization of the basolateral membrane with 100 µM nystatin was preliminarily confirmed by basolateral application of bumetanide, an inhibitor of the Na+-K+-2Cl- cotransporter located on the basolateral membrane of Calu-3 cells, and a bumetanide-sensitive component was not detected 30 min after addition of nystatin (data not shown). The 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB)-sensitive component of ICl (NPPB-sensitive ICl) reflects the apical Cl- permeability through CFTR in Calu-3 cells because the only Cl- channels detected on the apical membrane of Calu-3 cells are CFTR channels (11, 16). In this basolateral solution, CaCl2 was increased to 4 mM to compensate for the Ca2+-chelating capacity of the gluconate (17). On the other hand, the K+ current across the basolateral membrane (IK) was estimated after permeabilization of the apical membrane with nystatin (50 µM) for more than 30 min and establishment of an apical-to-basolateral K+ concentration gradient (12, 15). Complete permeabilization of the apical membrane with 50 µM nystatin had been confirmed by apical application of phlorizin, an inhibitor of the Na+-glucose transporter located on the apical membrane of this cell line (18). Apical NaCl was replaced by equimolar K-gluconate, while basolateral NaCl was substituted with equimolar Na-gluconate. Cl- was removed from these solutions. On the basolateral membrane, the major K+ conductance was produced by the hIK-1 channel (12, 13).

Measurement of Intracellular Ca2+ Concentration
Unlike previous studies, we grew Calu-3 cells as a polarized monolayer on permeable membrane supports (Transwell inserts, Clear type; Costar) to measure [Ca2+]i in response to extracellular ATP from the apical and basolateral surfaces separately. This culture strategy yields an airway epithelial morphology that closely mimics that in vivo (19). In addition, this culture system permits access of reagents to both epithelial aspects. 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 thereafter 0.5 and 1 ml PSS were added to the apical and basolateral membranes, respectively. Fluorescence signals were collected for 20 ms at 4- to 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 adding 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 our preliminary experiments, ionomycin (1 µM) added either to the apical or basolateral compartment caused a good increase in [Ca+]i, indicating that the [Ca2+]i measurement system allowed sufficient reagents to reach either surface of the membrane (data not shown).

Reagents
ATP, ADP, UTP, UDP, ATP{gamma}S, ADPßS, MRS-2179, nystatin, SQ22536, PPADS, D609, U73122, suramin, chelerythrine, 1,2-bis (o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM), and NPPB were obtained from Sigma-Aldrich. Charybdotoxin was purchased from Peptide Institute Inc. (Osaka, Japan). ET-18-OCH3 and Gö6983 were products of Calbiochem (San Diego, CA). Nystatin stock solution (100 mM) was made and sonicated for 30 s just before use.

Analysis of Results
Concentration–response curves in the present study were obtained using the computer program Cricket Graph version 1.5.3 for Macintosh (Computer Associates International Inc., Islandia, NY). All data are expressed as means ± SEM with the number of experiments used (n). Comparisons between two groups were made by unpaired Student's t test, and those among multiple groups were performed by one-way ANOVA. P < 0.05 was considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differences between Apical and Basolateral ATP-induced Isc
The basal Isc and Rt measured just before addition of ATP were 12.2 ± 0.5 µA/cm2 and 497.1 ± 15.8 {Omega}cm2 (n = 71), respectively. The Isc responses after addition of apical and basolateral ATP differed markedly in Calu-3 human airway epithelial cells. Namely, application of ATP (100 µM) to the apical face led to a rapid increase in Isc reaching a peak in 1–3 min followed by a gradual Isc decay (Figure 1A). The peak value from the baseline ({Delta} Isc) was 45.3 ± 2.5 µA/cm2 (n = 45), and the value of {Delta} Isc was reduced to 17.7 ± 1.1 µA/cm2 (n = 45) at 30 min after addition of ATP. On the other hand, basolaterally applied ATP (30 µM) elicited a sharp transient increase in Isc, whose {Delta} Isc was 206.7 ± 17.1 µA/cm2 (n = 26), followed by a steep fall to the level just above the baseline (Figure 1B). The distinct reactions to ATP applications from either side were abolished by removal of Cl- from the extracellular solutions, suggesting that the responses are totally composed of Cl- secretion (data not shown).



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  		Figure 1. Difference in effects of apical and basolateral applications of ATP on short-circuit current (Isc) in cultured Calu-3 human airway epithelial cells. (A) Apical (Api) ATP (100 µM) induced a rapid increase in Isc followed by a gradual decay. (B) Basolateral (Baso) ATP (30 µM) elicited a sharp transient increase in Isc followed by a steep fall to just above the baseline. (C and D) Both responses were markedly prevented by preincubation for 30 min with a selective P2Y1 blocker MRS-2179 (100 µM) or other P2Y blockers like PPADS (100 µM) and suramin (100 µM), comparing the peak values from the baseline ({Delta}Isc) with the control. These P2Y1 blockers and ATP were added to the same compartment. (E) Concentration-dependent effects were observed due to either apical or basolateral ATP. Increases in Isc ({Delta} Isc) caused by apical ATP were smaller than those due to basolateral ATP. The {Delta} Isc were quantified by subtracting the basal Isc value that is just before addition of agonists from the peak values of the Isc. Data are means ± SEM (n = 4–45). C and D: #P < 0.0001, significantly different from the control values. E: Significant differences from the corresponding values with apical ATP are expressed with *P < 0.05, **P < 0.005, and ***P < 0.0001 (unpaired Student's t test).

 
A previous study using RT-PCR to detect subtypes of P2Y receptors reported that only P2Y1 mRNA is expressed in Calu-3 cells (3). As expected, the ATP-induced responses were interrupted by a selective P2Y1 inhibitor (MRS-2179 at 100 µM) or non-selective P2Y blockers (suramin or PPADS at 100 µM) (Figures 1C and 1D). This suggests the involvement of P2Y1-mediated signal transduction in apical and basolateral ATP-induced Cl- secretion. Figure 1E shows the concentration-dependent reactions of ATP-induced Isc. The EC50 values of {Delta} Isc were ~ 10 µM for apical and 20 µM for basolateral application of ATP. As is evident, the basolateral ATP-induced {Delta} Isc surpassed the apical one in spite of a much lower sustained component (see Figures 1A and 1B).

Figures 2A and 2B show the effects of extracellular ADP applied from either aspect of the monolayer. {Delta} Isc were 50.0 ± 2.3 µA/cm2 (n = 4) in apical ADP (100 µM) and 192.9 ± 23.5 µA/cm2 (n = 6) in basolateral ADP (30 µM), indicating that there was no significant difference between the effects of ATP and ADP. In contrast, no discernible response was produced by additions of UTP or UDP (Figures 2C–2F). Figure 3 shows the effects of nonhydrolysable nucleotides ADPßS and ATP{gamma}S on the Isc. Application of ADPßS or ATP{gamma}S to each compartment (100 µM to the apical and 30 µM to the basolateral side) evoked responses analogous to ATP and ADP. However, the effects of ADPßS were more potent than those of ATP{gamma}S. Namely, when ADPßS and ATP{gamma}s were applied to the apical side, the stimulated {Delta} Isc were 35.5 ± 4.7 µA/cm2 (n = 4) and 11.4 ± 3.5 µA/cm2 (n = 4, P < 0.01), respectively. When basolaterally applied, these values were 117.2 ± 16.6 µA/cm2 (n = 4) and 65.7 ± 9.0 µA/cm2 (n = 6, P < 0.05), respectively.



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  		Figure 2. Representative recordings of Isc responses induced by ADP (A, B), UTP (C, D), and UDP (E, F). Apical (Api, 100 µM) and basolateral (Baso, 30 µM) applications of ADP mimicked the responses to ATP. Note that UTP or UDP applied to either side had no significant effect.

 


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  		Figure 3. Effects of nonhydrolysable nucleotides ADPßS (A and B) and ATP{gamma}S (C and D) on the Isc. Although apical (Api, 100 µM) and basolateral (Baso, 30 µM) applications of these nucleotides evoked responses analogous to ATP and ADP (see Figures 1 and 2), the effects of ADPßS were more potent than those of ATP{gamma}S.

 
Coupling of the P2Y1 Receptor to Phospholipase C and Adenylate Cyclase
Because the type of P2Y receptor seems unlikely to differ between the apical and basolateral membranes, a difference in cytosolic signal transduction is a possibility. To test this hypothesis, ATP-induced responses were observed in the presence of G protein–coupled enzyme inhibitors. As shown in Figures 4A and 4B, Isc responses to apically and basolaterally applied ATP were unaffected by pretreatment with the phosphatidylinositol-phospholipase C (PI-PLC) inhibitors U73122 (100 µM) or ET-18-OCH3 (100 µM, data not shown). In the presence of U73122, the {Delta} Isc induced by apical and basolateral applications of ATP were 41.8 ± 2.4 µA/cm2 (n = 6) and 178.4 ± 22.0 µA/cm2 (n = 4), respectively. In contrast, the phosphatidylcholine-phospholipase C (PC-PLC) antagonist D609 (100 µM) markedly prevented each ATP-induced response (Figures 4C and 4D). In the presence of this antagonist, the {Delta} Isc due to apical and basolateral applications of ATP were decreased to 4.5 ± 0.6 (n = 4, P < 0.0001) and 31.1 ± 9.7 µA/cm2 (n = 4, P < 0.001), respectively. On the other hand, preincubation with SQ22536 (1 mM, an adenylate cyclase inhibitor) also significantly inhibited Isc in response to ATP on either side; the {Delta} Isc by apical and basolateral ATP were 16.9 ± 2.5 (n = 5, P < 0.001) and 73.3 ± 22.7 µA/cm2 (n = 4, P < 0.01), respectively (Figures 4E and 4F).



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  		Figure 4. Effects of G protein–coupled enzyme inhibitors of PI-PLC (U-73122, 100 µM; A and B), PC-PLC (D609, 100 µM; C and D), and adenylate cyclase (SQ22536, 1 mM; E and F) on apical (Api, 100 µM) and basolateral (Baso, 30 µM) ATP-induced Isc. Cells were bilaterally pretreated with U-73122 for 4 h, or with D609 and SQ22536 for 30 min.

 
Ca2+ Dependency of the ATP-Induced Responses
The PC-PLC–mediated signaling pathway is involved in the formation of diacylglycerols (DAG), whose main physiologic objective is activation of the Ca2+ mobilization pathway and protein kinase C (PKC). To examine the involvement of cytosolic Ca2+ in P2Y1-mediated signal transduction, ATP-induced Isc were followed in the presence of BAPTA-AM (20 µM, bilateral) or ChTx (100 nM, basolateral), resulting in significant reduction by these agents (Figures 5A and 5B). In contrast, pretreatment with PKC inhibitors, chelerythrine (10 µM) or Gö 6,983 (1 µM), for 30 min was without effect on the ATP-induced responses (Figures 5A and 5B). Figures 5C and 5D are representative recordings of [Ca2+]i in response to apical and basolateral ATP. Interestingly, [Ca2+]i was raised in an oscillatory fashion by the apical application of ATP whereas little change was detected due to the application of basolateral ATP (Figures 5C and 5D).



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  		Figure 5. Dependence of ATP-induced responses on cytosolic Ca2+ and protein kinase C (PKC). Pretreatment with BAPTA-AM (20 µM, bilateral) or charybdotoxin (ChTx, 100 nM, basolateral), but neither chelerythrine (10 µM, bilateral) nor Gö6983 (1 µM, bilateral), significantly inhibited the {Delta}Isc raised by ATP applied to apical (A, Api, 100 µM) and basolateral (B, Baso, 30 µM) side. Data are means ± SEM (n = 4–45). *P < 0.01, **P < 0.001, significantly different from the control values (unpaired Student's t test). (C and D) Representative recordings of cytosolic Ca2+ concentrations ([Ca2+]i) in response to apical and basolateral ATP estimated by fluo-3 signals. Note that oscillatory elevation of [Ca2+]i was observed by the application of apical (C) but not basolateral ATP (D).

 
Separate Measurements of Apical ICl and Basolateral IK
Figure 6 shows separate measurements of apical ICl and basolateral IK in response to extracellular ATP (apically at 100 µM and basolaterally at 30 µM) in nystatin-permeabilized monolayers. Each application of ATP stimulated similar inward sustained ICl (Figures 6A and 6C). Values of the NPPB (a CFTR blocker)-sensitive ICl in the sustained component of the ATP-induced Isc were 27.6 ± 10.2 (n = 5, in apical ATP) and 35.3 ± 6.6 µA/cm2 (n = 10, in basolateral ATP), which were totally abrogated by the pretreatment with SQ22536 (1 mM, Figures 6B and 6D). On the other hand, extracellular ATP applied from each aspect generated different patterns of outward IK that were prevented by basolateral ChTx (Figures 6E–6H). The values of the {Delta} IK due to apical (13.1 ± 1.3 µA/cm2, n = 7; Figure 6E) and basolateral ATP (48.4 ± 3.5 µA/cm2, n = 6; Figure 6G) were suppressed to 2.8 ± 0.3 µA/cm2 (n = 5, P < 0.05; Figure 6F), and 9.2 ± 0.4 µA/cm2 (n = 4, P < 0.05; Figure 6H), respectively, by ChTx. It is noted that the changes in IK correlated well to that of Isc under ATP-stimulated conditions (see Figure 1).



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  		Figure 6. (A–D) Effects of extracellular ATP on apical membrane Cl- current (ICl) after establishment of an apical to basolateral Cl- gradient and permeabilization of the basolateral membrane with nystatin (100 µM). Apical (Api, A) or basolateral (Baso, C) ATP-induced ICl, which is blocked by SQ22536 (1 mM, bilateral, B and D), was inhibited by the application of NPPB (100 µM, bilateral), a CFTR blocker. SQ22536 was applied 30 min before the ATP addition. (E–H) Effects of extracellular ATP on basolateral membrane K+ current (IK) after establishment of an apical to basolateral K+ gradient and permeabilization of the apical membrane with nystatin (50 µM). Note that apical (E) and basolateral ATP (G) induced different patterns of IK that were inhibited by application of charybdotoxin (ChTx, 100 nM, basolateral, F and H) 10 min before ATP addition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several types of P2Y have been widely implicated in anion secretion in airway epithelia in vivo and in vitro (20, 21). The present study demonstrated that access of extracellular ATP to the apical and basolateral membranes generates Cl- secretions with different properties in human airway epithelial Calu-3 cells. The apical ATP evoked an immediate rise in Isc followed by a gradual decay whereas the basolateral ATP caused a transient rise (< 2 min) with a peak ~ 4-fold higher than the apical one. However, the evident differences between the apical and basolateral ATP actions could not be explained by different P2Y subtypes. Indeed, both apical and basolateral ATP-induced responses were blocked by the same P2Y1 antagonists (Figure 1), in agreement with previous data using RT-PCR techniques (3). Of the five mammalian P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11), the absence of P2Y2, P2Y4, and P2Y6 on the bilateral surfaces of Calu-3 cells was certified by the pharmacologic profiles of the Calu-3 cell monolayer, which failed to respond to UTP and UDP (see Figure 2). In addition, P2Y11 (human) is more sensitive to ATP{gamma}S than to ATP, and ADP is an ineffective agonist for this receptor (2, 22). The pharmacologic properties of P2Y11 are not consistent with the results in Figures 2 and 3. It is established that ADP, ATP, and ADPßS are effective ligands for the P2Y1 receptor, which is not activated by UDP or UTP. This is in agreement with the pharmacologic properties of Calu-3 demonstrated in the present study. Thus, it is highly possible that the P2Y1-receptor or P2Y1-like receptors are responsible for extracellular ATP-mediated signal transduction both on the apical and basolateral membrane.

In all cells, biological responses to extracellular nucleotides are complicated not only by the presence of various purinergic receptors but also by the rapid ATP catabolism that is caused by ectonucleotidases located on the cell surface (23, 24). High-performance liquid chromatography analysis of nucleotides revealed that liquid sampled from the undisturbed apical surface of Calu-3 epithelia contains ATP, ADP, AMP, and adenosine, of which ADP is most predominant (25). This report suggests that ecto-ATPase–induced conversion from ATP to ADP is the most frequent and highest activity on the apical surface of Calu-3 cells, although no information is available regarding basolateral ectonucleotidases. However, heterogeneous expression of ectonucleotidases could not account for the differential actions of ATP/ADP because the nonhydrolysable nucleotides ATP{gamma}S and ADPßS mimicked both apical and basolateral ATP/ADP-induced responses. In addition, the greater potency of ADPßS over ATP{gamma}S suggests that bilateral ATP exerts its actions, in part, after converting to ADP on the bilateral cell surface of the polarized Calu-3 monolayer.

According to previous reports, P2Y1 is believed to be associated with Gq/11-coupled PLC (2). PLC is classified into two types: PI-PLC and PC-PLC (26). PI-PLC–mediated phospholipid hydrolysis had previously been thought to be the sole mechanism for the production of DAG, which triggers Ca2+ mobilization and PKC activation (27). There are several lines of evidence that P2Y-mediated Ca2+ mobilization is coupled to PI-PLC (2, 23). However, recent investigations have shed light on PC-PLC as an alternative pathway for production of DAG (26). In polarized Calu-3 cells, as shown in Figure 4, apical and basolateral ATP-induced Isc were markedly interrupted by pretreatment with D609, a specific PC-PLC inhibitor, but not by U73122 or ET-18-OCH3, PI-PLC inhibitors. To ensure the results, we extended the exposure time of these PI-PLC inhibitors from 30 min to 4 h, resulting in no significant effect. Thus, these results suggest the possible coupling of P2Y1 receptors with PC-PLC on each membrane. P2Y1-like receptor–mediated activation of PC-PLC has previously been reported in endothelial cells, although the activation may be caused downstream of PKC (28). Nevertheless, the present study showed that PKC inhibitors like chelerythrine and Gö6983 fail to affect ATP-induced responses, suggesting the direct coupling of P2Y1 with PC-PLC and little contribution of PKC to P2Y1-mediated anion secretion (see Figure 5).

The present data show that ATP-induced responses were attenuated not only by a PC-PLC inhibitor but also an adenylate cyclase inhibitor SQ22536, suggesting the bilateral presence of dual coupling to PC-PLC and adenylate cyclase in the P2Y1 receptor on Calu-3 cells. Similar data have been reported for other types of mammalian cells. In mouse ventricular myocytes predominantly expressing P2Y1, stimulation by ATP simultaneously increases cytosolic PKC and adenylate cyclase–cAMP-PKA activity through heterotrimeric G protein (29). Recent studies have found that ATP promotes cAMP formation via P2Y coupled to adenylate cyclase in newborn rat type II alveolar cells that express P2Y1 (2, 30). The P2Y11 receptor, which has 33% amino acid identity with the P2Y1 receptor, is dually coupled to both PLC and adenylate cyclase stimulation (31). Both the PC-PLC–mediated Ca2+ mobilization and adenylate cyclase–mediated cAMP formation are implicated in transepithelial anion transport via different signal transductions (12).

Although it is well known that apical CFTR is essential as a Cl- export pathway in Calu-3 cells, basolateral K+ channels, whose activity contributes to the driving force for CFTR-mediated Cl- secretion, also play an important role (12, 15). Cowley and Linsdell (32) have reported that Calu-3 cells express the K+ channel genes KCNN4 (hIK-1 channel), KCNQ1 (KvLQT1), KCNE2, and KCNE3. They concluded that a KCNQ1-containing channel complex, such as KCNQ1-KCNE3, may be involved in cAMP-activated K+ conductance. However, several lines of evidence have shown that 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, could be identified in the permeabilized Calu-3 monolayer (12, 32). Thus, an increase in [Ca2+]i stimulates Cl- secretion via activation of the hIK-1 channels especially, whereas an increase in cytosolic cAMP concentration ([cAMP]i) activates apical CFTR via protein kinase A (PKA)-dependent mechanisms. If CFTR were activated without opening any basolateral K+ channels, Cl- secretion would subside after transient responses because the equilibrium potential of Cl- is generally close to the resting potential on the apical membrane (12). The present study showed that apical and basolateral ATP-induced Isc were diminished in the presence of a cytosolic Ca2+ chelator (BAPTA-AM) and a selective hIK-1 channel blocker (ChTx). These data suggest that PC-PLC–mediated [Ca2+]i elevation and the resultant hIK-1 channel activation are involved in bilateral P2Y1-mediated Cl- secretion in addition to adenylate cyclase–cAMP-PKA–dependent mechanisms. The Ca2+ dependency of the P2Y1-mediated responses naturally prompted us to anticipate that ATP applied from either side would increase the [Ca2+]i. Unexpectedly, however, fluo-3 signal measurement revealed that apical ATP induced intracellular Ca2+ responses in an oscillatory fashion, whereas basolateral ATP failed to do so. The cause of the unilateral [Ca2+]i mobilization could be explained by the previous reports using other epithelial cells. In pancreatic epithelial cells, an agonist-induced increase in [Ca2+]i is initiated at the apical pole of the cell and is rapidly propagated as a Ca2+ wave toward the basolateral region (33, 34). Ashby and coworkers (35) have demonstrated that local uncaging of caged Ca2+ in the apical granule-containing region of pancreatic acinar cells can trigger cytosolic Ca2+ waves, which spread from the apical region toward the basal membrane, whereas local Ca2+ uncaging in the basolateral region consistently failed to initiate Ca2+ wave. The unidirectional Ca2+ spread is due to the differences between apical and basolateral poles in the distribution of cytosolic Ca2+ store sites (36). The cytosolic Ca2+ wave also occurs in a variety of cell types including airway epithelial cells (4, 37). Considering these reports, we hypothesize that localized Ca2+ rising in the subcellular regions of either membrane due to P2Y1 stimulation may spread as a Ca2+ wave only from the apical to the basolateral pole. Because our experimental system could detect only whole cell [Ca2+]i changes, local transient Ca2+ rises adjacent to the basolateral membrane in response to the basolateral ATP might not be observed in spite of transient activation of the basolaterally-located hIK-1 channel. However, this hypothesis was not directly tested in the present study, and further studies are needed to clarify this point. At any rate, data from fluo-3 signal measurements suggest differential changes in [Ca2+]i, and the resultant activation of the basolateral hIK-1 channel may be relevant to the differential ATP actions. To confirm this point, separate measurements of apical membrane ICl and basolateral membrane IK were conducted in the monolayer permeabilized from each side. Apical ICl measured under the basolateral permeabilized condition reflecting CFTR-mediated Cl- conductance were continuously potentiated by the application of ATP from either side, and these responses were prevented by the pretreatment with SQ22536, as observed in the nonpermeabilized monolayer (see Figure 4). The patterns of the sustained ICl generated by the stimulation of apical and basolateral P2Y1 did not evidently correlate with that of Isc. Instead, there is a good correlation between ATP-induced Isc and IK, suggesting that the hIK-1 channel is a rate controller for bilateral ATP-induced Cl- secretion.

The finding that the adenylate cyclase inhibitor, SQ22536, reduces the response to ATP might indicate that the responses are partially mediated by the actions of ATP metabolite adenosine on the A2B receptor that is associated with GS-coupled adenylate cyclase (25). In our preliminary experiments to rule out this possibility, Calu-3 monolayer generated anion secretion in response to adenosine (10 µM) applied to apical but not basolateral side, and the response was unaffected by MRS-2179, a selective P2Y1 antagonist, which completely interrupted ATP-induced Isc (see Figure 1). These observations suggest that ATP-induced responses do not include A2B receptor–mediated components. Nevertheless, responses to ATP and ADPßS, which were applied to the apical but not to the basolateral solution, were partially inhibited by A2B receptor antagonists, such as alloxazine and 8-(p-sulfophenyl) theophylline, in Calu-3 cells (data not shown). In rat heart, ADPßS-induced responses are similarly inhibited by the A2B receptor antagonists (38). In spite of these complicated data, recent investigations have demonstrated that the A1 and the P2Y1 receptor, both of which are colocalized in the overlap regions, form a heterometric oligomerization that exerts novel pharmacologic and functional characteristics (39). Further, other groups have suggested the presence of adenosine receptor–like P2Y1 receptors in rat type II cells and bovine airway epithelial cells (6, 30). Thus, on the apical membrane of Calu-3 cells where A2B and P2Y1 receptor are colocalized, heterometric assembly of these two receptors might form A2B-like P2Y1 receptors, although this hypothesis is not tested in the present study. Strictly speaking, therefore, extracellular ATP-sensitive receptors on the apical and basolateral membrane may not be completely identical.

Overall, Calu-3 cells possess the P2Y1 receptor on both the apical and basolateral membranes, but when activated by nucleotides, apical and basolateral receptors evoked different patterns of Cl- secretion via similar signal transduction pathways that involve both PC-PLC and adenylate cyclase. The difference appears to be due to distinct behaviors of intracellular Ca2+ and the resultantly activated hIK-1 channel on the basolateral membrane (Figure 7). Namely, the Cl- secretory rates after bidirectional P2Y1 stimulations are both regulated by the hIK-1 channel–mediated K+ conductance. This indicates the importance of this channel in airway mucociliary clearance regulated by extracellular nucleotides.



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  		Figure 7. A hypothesized scheme of regulatory pathways of anion transport mediated by apical and basolateral P2Y1 receptors; (+) indicates stimulatory effects. The P2Y1 receptors on both sides couple with both PC-PLC and adenylate cyclase (AC), leading to Cl- secretion through simultaneous activation of CFTR-mediated ICl and hIK-1 channel–mediated IK. Because the equilibrium potential of Cl- is generally close to the resting potential on the apical membrane, activation of the hIK-1 channel is crucial for driving Cl- export across the apical membrane. The AC-mediated pathway linking to the cAMP-PKA transduction activates the cAMP-dependent Cl- channel CFTR on the apical membrane. On the other hand, the PC-PLC–mediated signaling pathway links to the formation of diacylglycerols (DAG) from phosphatidylcholine (PC), whose main physiologic objective is Ca2+ mobilization. Through this pathway, apically applied ATP increases [Ca2+]i of the whole cell whereas basolaterally applied ATP transiently increases only the localized Ca2+ level adjacent to the hIK-1 channel, resulting in anion secretions with different properties.

 

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

Received in original form May 7, 2003

Received in final form August 2, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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