Ion transport regulated by protease-activated receptor 2 in human airway Calu-3 epithelia

Abstract

1. We examined the mechanisms underlying anion secretion mediated by protease-activated receptor 2 (PAR2) and its role in the regulation of ion transport, using polarized human airway Calu-3 cells.

2. PAR2 stimulation by trypsin and a PAR2-activating peptide (PAR2AP), especially from the basolateral aspect, caused transient Cl⁻ secretion due to cytosolic Ca²⁺ mobilization.

3. Antagonists of PI-PLC (U73122, ET-18-OCH₃) and inositol 1,4,5-triphosphate (xestospongin C (Xest C)) were without effect on the PAR2AP-mediated Cl⁻ secretion, whereas it was attenuated by D609 (a PC-PLC inhibitor) and phorbol 12-myristate 13 acetate (PMA, a PKC activator).

4. Even 30 min after removal of PAR2AP after a 10-min-exposure, cells were still poorly responsive to PAR2 stimulation, but the reduced responsiveness was upregulated by a PKC inhibitor, GF109203X (GFX).

5. Pretreatment with PAR2AP did not affect responses to anion secretagogues, such as isoproterenol, forskolin, thapsigargin, 1-ethyl-2-benzimdazolinone, and adenosine, but ATP-induced responses were significantly reduced. Nystatin permeabilization studies revealed that the presence of PAR2AP prevented ATP-induced increments in basolateral membrane K⁺ conductance without affecting apical membrane Cl⁻ conductance.

6. ATP-elicited Ca²⁺ mobilization, which was sensitive to D609 and PMA, was inhibited by the pretreatment with PAR2AP, and this inhibition was blunted by the presence of GFX.

7. Collectively, stimulation of PAR2 generates a brief response of Cl⁻ secretion through PC-PLC-mediated pathway, followed by not only auto-desensitization of PAR2 itself but also cross-desensitization of a PC-PLC-coupled purinoceptor. The two types of desensitization seem likely to have PKC-mediated downregulation of PC-PLC in common.

Introduction

Protease-activated receptors (PARs) belong to a family of seven transmembrane G protein-coupled receptors that are activated by extracellular proteases (Macfarlane et al., 2001). These receptors are activated by the proteolytic unmasking of a receptor-bound, NH₂-terminal-tethered ligand domain, which is then able to interact with the extracellular loop of the receptor itself and initiate signaling. To date, four PARs (PAR1–4) have been characterized (Macfarlane et al., 2001). Thrombin activates PAR1, -3, and -4, whereas trypsin and mast cell tryptase activate PAR2 and possibly PAR4 (Macfarlane et al., 2001). Synthetic peptides based on the receptor-activating sequence of the tethered ligand can directly bind and activate the receptors (Dery et al., 1998). Various branches of science have been paying growing attention to the functional relevance of PARs, because of their systemic distribution and potential roles in numerous systems, including homeostasis, inflammation, and the regulation of smooth muscle tone (Dery et al., 1998; Cocks et al., 1999; Cocks & Moffatt, 2001; Macfarlane et al., 2001). PAR2 is the PAR predominantly expressed in human airway epithelia (Cocks & Moffatt, 2000). Recently, upregulation of PAR2 in the respiratory epithelium of patients with bronchial asthma was reported (Knight et al., 2001). Further, Northern blotting analysis of human tissues shows that trypsin-like protease mRNA is most prominently expressed in tracheal tissue, suggesting that this protease is localized in the airway (Yamaoka et al., 1998). These reports implicate PAR2 in the pathophysiology of chronic obstructive airway diseases.

It is well known that the chronic obstructive airway diseases share aspects of mucous congestive diseases (Kellerman, 2002), in which excessive and tenacious mucus secretion causes airway obstruction and is involved in the morbidity and mortality of these diseases (Lundgren & Shelhamer, 1990; Aikawa et al., 1992). In particular, bronchial gland cells predominantly contribute to airway electrolyte secretion, which is accompanied by fluid movement, thereby forming low viscosity mucus to maintain efficient mucociliary clearance (Shimura, 2000). Despite the significance of the electrolyte transport system in airway conductivity, little is known regarding the roles of PAR2 in this system. Thus, the aim of the present study was to establish how Cl⁻ secretion is regulated by PAR2-mediated signals, using polarized Calu-3 epithelia, which is a model of human airway submucosal gland serous cells (Devor et al., 1999). Through the investigations, we also found that PAR2 stimulation caused not only auto-desensitization but also cross-desensitization of a purinoceptor that is well known as a key molecule promoting mucociliary clearance in the airway (Ramminger et al., 1999; Inglis et al., 2000; Ito et al., 2004a).

Methods

Cell culture

Calu-3 human airway cells (American Type Culture Collection, Manassas, VA, U.S.A.) 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, U.S.A.) containing 10% fetal bovine serum (Invitrogen), 100 μg/ml streptomycin, and 100 U/ml penicillin (Invitrogen). The cells were maintained in tissue-culture flasks (T75) at 37°C in a 95% air–5% CO₂ humidified incubator. After reaching 80–90% confluence, cells were detached using a solution of 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, U.S.A.) 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, U.S.A.). 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 (Shen et al., 1994). The cells were fed by replacement of the basolateral medium every 48 h. Experiments were carried out after 7–13 days in culture.

Normal human bronchial epithelial cells (NHBE; BioWhittaker, Walkersville, MD, U.S.A.) seeded into T75 flasks were grown for detection of PAR2 mRNA in bronchial epithelial cell growth medium (BEGM; BioWhittaker) supplemented with bovine pituitary extract (52 μg ml⁻¹), hydrocortisone (0.5 μg ml⁻¹), human recombinant epidermal growth factor (0.5 ng ml⁻¹), epinephrine (0.5 μg ml⁻¹), transferrin (10 μg ml⁻¹), insulin (5 μg ml⁻¹), retinoic acid (0.1 μg ml⁻¹), triiodothyronine (6.5 μg ml⁻¹), gentamycin (50 μg ml⁻¹), and amphotericin B (50 μg ml⁻¹). The medium was changed every 48 h until cells were 90% confluent.

RT–PCR for detection of PAR2 mRNA

Total RNA was extracted from Calu-3 and NHBE cells, which had been grown to near confluence on T75 flasks, using Trizol reagent (Invitrogen). First-strand cDNA was generated using SuperScript III (Invitrogen) and random primers (Takara Bio, Shiga, Japan) according to the manufacturer's instructions. Reverse transcriptase–polymerase chain reaction (RT–PCR) was performed with a primer set of PAR2 forward 5′-CCTGAGTGGCACCATCC and PAR2 reverse 5′-CAGGGAGATGCCAATGGC. The size of the expected PCR product was 527 bp. PCR conditions consisted of 5 min at 97°C, followed by 40 cycles of 30 s of denaturing at 96°C, 30 s of annealing at 57°C, 30 s of extension at 72°C, with a final extension at 72°C for 10 min. The PCR product was visualized by loading a 10 μl sample on a 2% agarose gel.

Ussing-chamber experiments

Snapwell inserts on which Calu-3 cells had grown confluent were rinsed with physiological saline solution (PSS) and transferred to the modified Ussing chambers (EasyMount Chamber; Physiologic Instruments, San Diego, CA, U.S.A.) that contained PSS at 37°C. The PSS 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) using NaOH before addition of NaHCO₃. The pH of the solution was kept at pH 7.4 when gassed with a mixture of 5% CO₂ and 95% O₂. Transepithelial potential differences (PD) from the basolateral to the apical side of the monolayer were continuously measured under open-circuit conditions by a high-impedance millivoltmeter that could function as a voltage clamp with automatic fluid resistance compensation (VCC MC2, Physiologic Instruments). Transepithelial conductance (G_t) was determined by imposing short (0.5 s) current pulses (ΔI=2–10 μA), and the corresponding changes in transepithelial voltage (ΔV) were calculated using Ohm's law (G_t=ΔI ΔV⁻¹). The equivalent short-circuit current (I_eq) was calculated as G_t PD. I_eq represents net charge movement across the monolayer.

Permeabilized monolayers

To investigate apical membrane Cl⁻ conductance, the basolateral membrane was permeabilized with the pore-forming antibiotic nystatin (100 μM) for more than 30 min. This level of nystatin was determined as the concentration at which bumetanide, an inhibitor of the basolateral Na⁺–K⁺–2Cl⁻ cotransporter, had no effect on the ion current (Son et al., 2004). This procedure avoids the complexities associated with basolateral ion transporters and permits analyses of apical membrane Cl⁻ conductance (Devor et al., 1999; Ito et al., 2002; Son et al., 2004). Apical membrane Cl⁻ conductance was estimated as apical membrane Cl⁻ current (I_Cl) in the apical-to-basolateral Cl⁻ concentration gradient under closed-circuit conditions. The Cl⁻ concentration gradient was established by replacing NaCl with equimolar Na-gluconate in the basolateral PSS. I_Cl reflects the apical Cl⁻ permeability through cystic fibrosis transmembrane conductance regulator (CFTR) in Calu-3 cells because the apical anion conductance is almost completely derived from CFTR channels in Calu-3 cells (Haws et al., 1994; Devor et al., 1999). To completely eliminate the component of basolateral K⁺ conductance in this I_Cl measurement, these experiments were performed in the presence of clotimazole, an inhibitor of the human intermediate conductance Ca²⁺-activated K⁺ (hIK-1) channel. In this basolateral solution, CaCl₂ was increased to 4 mM to compensate for the Ca²⁺-chelating capacity of the gluconate.

The basolateral membrane K⁺ conductance was estimated by measuring the basolateral membrane K⁺ current (I_K) after permeabilization of the apical membrane with nystatin (50 μM) for 30 min and establishment of an apical-to-basolateral K⁺ concentration gradient (Devor et al., 1999; Son et al., 2004). Complete permeabilization of the apical membrane with 50 μM nystatin was confirmed by apical application of phlorizin, an inhibitor of the Na⁺-glucose transporter located on the apical membrane of this cell line (Ito et al., 2001; Son et al., 2004). Apical NaCl was replaced by equimolar K-gluconate, while basolateral NaCl was substituted with equimolar Na-gluconate. Cl⁻ was removed from these solutions to completely eliminate the component of apical Cl⁻ conductance. On the basolateral membrane, the major K⁺ conductance was produced by the hIK-1 channel in Calu-3 cells (Devor et al., 1999; Cowley & Linsdell, 2002).

Cytosolic Ca²⁺ mobilization assay

Calu-3 cells grown on the porous membranes of Transwell inserts (Costar) 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, U.S.A.). 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 (F_max) was obtained by addition of 10 μM ionomycin, and the minimum signal (F_min) was obtained by adding 10 mM EGTA to the cell monolayer. The cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was calculated according to the following formula:

in which the K_d was assumed to be 390 nM (Minta et al., 1989). In the present study, maximum [Ca²⁺]_i changes (Δ[Ca²⁺]_i) were compared between groups.

Chemicals

Forskolin, isoproterenol, 1-ethyl-2-benzimdazolinone (1-EBIO), bumetanide, clotrimazole, ATP, ADPβS, adenosine, D609, nystatin, trypsin, thrombin, thapsigargin, U73122, and GF109203X were obtained from Sigma-Aldrich Co. Xestospongin C and ET-18-OCH₃ were purchased from Calbiochem (La Jolla, CA, U.S.A.). Soybeans trypsin inhibitor was from Worthington (Lakewood, NJ, U.S.A.). SLIGKV-NH₂ (a PAR2-activating peptide: PAR2AP), was a product of Bachem (Bubendorf, Switzerland). VKGILS-NH₂ (a reverse sequence peptide of PAR2AP: revPAR2AP) was synthesized by Sawady Technology Co., Ltd (Tokyo, Japan). Stock solutions of ATP, ADPβS, trypsin, thrombin, and the soybean trypsin inhibitor were prepared by dissolution in distilled water. All other drugs were dissolved in DMSO. Nystatin stock solution (100 mM) was made and sonicated for 30 s just before use.

Data analysis

Numerical data are presented as means±s.e.m., where n refers to the number of experiments. Statistical differences were determined by Student's t-test for comparison of data between two groups or one-way analysis of variance for multiple group comparison. A value of P<0.05 was considered statistically significant.

Results

Bioelectric responses to PAR-related activators

Previous investigations have found that PAR2 are unilaterally located on the basolateral membrane in native airway epithelial tissues (Kunzelmann et al., 2002) and cultured bronchial epithelial cells (Danahay et al., 2001). Thus, we first applied various PAR activators to the basolateral face of the Calu-3 monolayer (Figure 1). Application of a PAR2, -4 activator, trypsin (50 U ml⁻¹), led to a rapid increase in apical side-negative PD from the baseline (−5.0±0.2 mV, n=41) to a peak (−15.8±0.5 mV), followed by a decay back to the baseline in a couple of minutes (Figure 1a). The corresponding values of I_eq are shown in Figure 1b. The peak values of I_eq (54.2±1.8 μA cm⁻²) minus basal ones (11.4±0.3 μA cm⁻²), which are expressed as ΔI_eq, were 42.8±1.8 μA cm⁻². The effects of trypsin (basolateral) were interrupted by the basolateral presence of a soybean trypsin inhibitor (data not shown). In contrast to the trypsin-induced responses, thrombin (50 U ml⁻¹), a PAR1, -3, -4 activator, failed to induce any bioelectric effect on the monolayer (Figure 1c and d), suggesting expression of PAR2 exclusively on the basolateral membrane. The trypsin-induced responses were mimicked by PAR2AP: SLIGKV-NH₂, which is a short synthetic peptide with proteolytically revealed PAR2 sequences (Figure 1e and f). Basolateral application of PAR2AP (50 μM) induced a transient increase in apical side-negative PD (ΔPD=22.1±0.8 mV, n=77; Figure 1e), resulting in transient elevation of I_eq (ΔI_eq=147.0±6.9 μA cm⁻²; Figure 1f). Also, the trypsin-induced responses were almost completely suppressed in the presence of PAR2AP (ΔI_eq=5.5±0.8 μA cm⁻², n=4, P<0.001), suggesting that trypsin and PAR2AP share a common receptor. Further, PAR2 specificity in the bioelectric responses was confirmed by the finding that the reverse sequence peptide of PAR2AP (revPAR2AP, 100 μM) had no effect on the PD and I_eq (Figure 1g and h).

Figure 1.

Representative traces of bioelectric responses to PAR activators in Calu-3 monolayer. Potential differences from the basolateral to apical side (PD) were monitored under an open-circuit condition with applications of repeated short (0.5 s) current pulses (10 μA). From the changes in PD, the equivalent short-circuit current (I_eq) was caluculated by Ohm's law. The cells were exposed to trypsin (50 U ml⁻¹, a and b), thrombin (50 U ml⁻¹, c and d), a PAR2-activating peptide (PAR2AP, 50 μM, e and f), and the reverse sequence peptide of PAR2AP (revPAR2AP, 50 μM, g and h) from the basolateral side.

Compared with the responses to basolateral trypsin, those to apical trypsin were small (Figure 2a). The small change made by apical trypsin was not due to its leakage to the basolateral side because a soybean trypsin inhibitor (50 U ml⁻¹) applied to the basolateral solution made no significant difference in responses to apical trypsin at 50 U ml⁻¹: ΔI_eq=5.1±0.8 μA cm⁻² (n=6) without and 5.4±0.3 μA cm⁻² (n=4) with 50 U ml⁻¹ soybean trypsin inhibitor. Similarly, apically applied PAR2AP induced only a small effect on the responses (ΔI_eq=14.7±3.2 μA cm⁻², n=4, Figure 2b), as was the case of trypsin. Figure 2c and d show cytosolic Ca²⁺ changes induced by basolateral and apical PAR2AP applications, indicating the consistency of PAR2-mediated ion transport with Ca²⁺ mobilization.

Figure 2.

Comparison of effects of basolateral (Baso) and apical (Api) applications of PAR2 agonists, trypsin (a) and PAR2AP (b), at various concentrations. The ΔI_eq was obtained by calculating the difference between peak values after the addition of PAR2 agonists and values just before their addition. Data are means±s.e.m. (n=4). (c, d) Representative tracings of [Ca²⁺]_i changes during exposure to PAR2AP from the basolateral (c) and apical side of the monolayer (d).

The PAR2-mediated I_eq almost completely comprises the Cl⁻ secretory component because PAR2AP-induced responses were abolished by removal of Cl⁻ from the extracellular solutions (ΔI_eq=13.3±1.9 μA cm⁻², n=4) and the presence of basolateral bumetanide (50 μM), a Na⁺–K⁺–2Cl⁻ cotransport inhibitor (ΔI_eq=23.2±0.8 μA cm⁻², n=4).

Molecular evidence of PAR2 expression

To examine PAR2 mRNA expression in Calu-3 cells, we analyzed total RNA via RT–PCR, which was performed using primers specific to the sequence of human PAR2. A 527-bp fragment was obtained, which was verified to be a PAR2 fragment by sequencing (Figure 3). PAR2 expression was also detected in normal human airway epithelial cells (NHBE; see Figure 3).

Figure 3.

Products of RT–PCR of RNA isolated from Calu-3 human airway cell line and normal human bronchial epithelial cells (NHBE), using gene-specific primer pairs targeted to PAR2. A 527-bp fragment of PAR2 was obtained from these two types of cells with reverse transcription (RT) of total RNA (+) but not without RT (–).

Potential mechanisms underlying PAR2-induced Cl⁻ secretion

G protein-coupled phospholipase C (PLC) is a major upstream enzyme to cause [Ca²⁺]_i mobilization and diacylglycerol (DAG)-mediated PKC activation (Hurley & Grobler, 1997). To examine the involvement of PLC in the PAR2-mediated Cl⁻ secretion, PD changes in response to PAR2 activators were observed in the presence of specific PLC inhibitors. Compared with the control (Figure 4a), PAR2AP (50 μM)-induced bioelectric responses in cells that had been exposed beforehand to phosphatidylinositol-phospholipase C (PI-PLC) inhibitors U73122 (100 μM Figure 4b) or ET-18-OCH₃ (100 μM) were still intact. Namely, ΔPD and ΔI_eq values generated by the application of PAR2AP in the control (22.3±1.4 mV and 162.3±19.0 μA cm⁻², n=7) were unaffected by U73122 (21.4±1.2 mV and 153.0±12.0 μA cm⁻², n=5) and ET-18-OCH₃ (22.4±1.6 mV and 163.6±26.5 μA cm⁻², n=5), respectively (Figure 4f). Further, PAR2-induced responses were also insensitive to (Xest C, 10 μM), a selective antagonist of inositol 1,4,5-triphosphate (Ins P₃; ΔPD=21.2±2.3 mV; ΔI_eq=140.5±21.2 μA cm⁻², n=5, Figure 4c). Similar pharmacological properties were reproduced in the cells stimulated by trypsin (data not shown). In contrast to these observations, pretreatment with a specific phosphatidylcholine-phospholipase C (PC-PLC) antagonist, D609 (100 μM), markedly prevented PAR2AP-mediated PD changes (ΔPD=3.9±0.5 mV, n=5) and the resultant I_eq (ΔI_eq=28.1±5.3 μA cm⁻², P<0.001), as demonstrated in Figure 4d. Figure 4e shows that the cells also became less responsive to PAR2AP in the presence of a PKC activator, PMA (1 μM): ΔPD=9.6±0.9 mV; ΔI_eq=51.0±2.8 μA cm⁻² (n=4, P<0.005). Summarized data of each ΔI_eq value is shown in Figure 4f.

Figure 4.

Mechanisms underlying PAR2-mediated anion secretion in Calu-3 cells. (a–e) Representative traces of potential differences from the basolateral to the apical side (PD) before and after application of the PAR2-activating peptide (PAR2AP, 50 μM, in the presence of the vehicle (0.1% DMSO: Control, a), U73122 (100 μM, b), xetospongin C (Xest C, 10 μM, c), D609 (100 μM, d), and PMA (1 μM, e). PD was monitored under open-circuit condition with applications of repeated short (0.5 s) current pulses (10 μA). (f) Summary of the PAR2AP-induced elevation of the equivalent short-circuit current (ΔI_eq), which was calculated by PD data. U73122, Xest C, D609, and PMA were applied 30 min prior to addition of PAR2AP. Data are means±s.e.m. (n=4–7). ^*P<0.005, ^**P<0.001.

Auto-desensitization induced by PAR2 activation

After completion of transient reactions due to PAR2AP (50 μM, basolateral), additional PAR2 stimulation (PAR2AP, 50 μM, basolateral) in the continuous presence of PAR2AP hardly generated responses (Figure 5a). The desensitization was not counteracted by pretreatment with the PKC inhibitor GFX (1 μM, Figure 5b). Figure 5c is a summary of the experiments shown in Figure 5a and b, indicating that the I_eq induced by the second PAR2AP application was 13.3±2.2% (n=4) of the first one in the control, and 11.3±1.4% (n=4) in the presence of GFX. When PAR2AP was removed after being present for 10 min, cells had only partially recovered their responsiveness to PAR2AP even 30 min later (Figure 5d), suggesting the desensitization remained for a long time after the wash-out. In contrast to the results shown in Figure 5a–c, however, the recovery in this case was further potentiated by the presence of GFX (Figure 5e): the responses to the second application was 43.9±3.5% (n=4) of the response to the first in the control and 71.1±3.4% (n=4, P<0.005) in the presence of GFX (Figure 5f).

Figure 5.

Auto-desensitization of PAR2-mediated signals in Calu-3 cells. (a–c) In the continuous presence of PAR2AP (50 μM, basolateral), I_eq responses to additional PAR2AP (50 μM, basolateral) were severely suppressed (a), and the suppression was not counteracted by the presence of the PKC inhibitor GF109203X (GFX, 1 μM, b). (c) Summary of the experiments shown in (a) and (b). Recovery rates (ordinates) were obtained by dividing the second ΔI_eq by the first one. (d–f) PAR2AP was removed after 10 min exposure, and PAR2AP was reapplied 30 min after its removal in the absence (d) and presence of GF109203X (GFX, 1 μM, e). (f) Summary of the experiments shown in (d) and (e). GF109203X was applied 30 min before the first application of PAR2AP. Data are means±s.e.m. (n=4). ^*P<0.005, significantly different from the control values.

Cross-desensitization caused by PAR2 activation

Next, we observed the effect of PAR2 stimulation on responses to various anion secretagogues. As shown in Figure 6a–d, I_eq responses to isoproterenol (10 nM, a β-adrenergic agonist), forskolin (10 μM, an adenylate cyclase (AC) activator), thapsigargin (1 μM, a Ca²⁺ mobilizing agent due to inhibition of Ca²⁺-ATPase and stimulation of capacitative Ca²⁺ entry), 1-EBIO (1 mM, a direct opener of human intermediated conductance, inward-rectifying Ca²⁺-activated K⁺ [hIK-1] channels) were unaffected by pretreatment with PAR2AP (50 μM, for 30 min). After PAR2 pretreatment, however, the cells became less responsive only to ATP (Figure 6e) and ADPβS. Namely, ΔI_eq produced by ATP (100 μM, apical) and ADPβS (100 μM, apical) were reduced from 46.6±3.7 μA cm⁻² (n=10) and 32.9±1.4 μA cm⁻² (n=19) to 16.6±1.5 μA cm⁻² (n=8, P<0.001) and 19.4±1.6 μA cm⁻² (n=4, P<0.001), respectively. In contrast, responses to an A_2B agonist, adenosine, a metabolite of ATP through ectonucleotidases, were intact in the presence of PAR2AP (Figure 6f).

Figure 6.

PAR2-induced desensitization of the subsequent heterologous responses. I_eq responses to isoproterenol (10 nM, basolateral, a), forskolin (10 μM, basolateral, b), thapsigargin (1 μM, apical, c), and 1-ethyl-2-benzimdazolinone (1-EBIO, 1 mM, bilateral, d) were unaffected by pretreatment with PAR2AP (PAR2AP (+), 50 μM, basolateral) applied 30 min prior to these anion secretagogues. In contrast, ATP-induced I_eq was inhibited by the presence this PAR2 agonist (e), although the responses to adenosine, a metabolite of ATP through ectonucleotidases, were intact under this condition (f). Data are means±s.e.m. (n=4–12).

It was previously reported that extracellular ATP and ADPβS cause Cl⁻ secretion via P2Y₁ purinoceptor (P2Y₁R), which is coupled both to AC and PC-PLC in Calu-3 cells (Communi et al., 1999; Son et al., 2004). Thus, there are three possible explanations for the attenuation of P2Y₁R-mediated Cl⁻ secretion: the stimulation of PAR2 inhibits (1) the AC/cAMP pathway relevant to CFTR activities, (2) the PC-PLC-Ca²⁺ pathway regulating the basolateral hIK-1 channel that provides a driving force for Cl⁻ secretion, or (3) both. To rule out the involvement of cAMP-dependent signal transductions in the PAR2-induced suppression of P2Y₁R signals, apical membrane Cl⁻ conductance, which reflects CFTR (a cAMP-dependent Cl⁻ channel)-mediated anion conductance, was followed by apical application of extracellular ATP (100 μM) under nystatin-permeabilized conditions (Figure 7a and b). However, the presence of PAR2AP (50 μM, Figure 7b) failed to affect the inward I_Cl across the apical membrane in the apical to basolateral Cl⁻ gradient (Figure 7b). Indeed, there was no significant difference in the ATP-augmented ΔI_Cl between the groups with PAR2AP (43.9±3.0 μA cm⁻², n=4) and without (41.7±4.4 μA cm⁻²). In contrast, an ATP-induced increase in basolateral membrane I_K (ΔI_K=28.2±0.7 μA cm⁻², n=4, Figure 7c), which is highly sensitive to charybdotoxin, a selective hIK-1 inhibitor (Son et al., 2004), was significantly reduced by the pretreatment with PAR2AP (ΔI_K=14.7±0.6 μA cm⁻², n=4, P<0.001, Figure 7d).

Figure 7.

(a, b) Effects of PAR2 activation on extracellular ATP-induced Cl⁻ conductance across the apical membrane in nystatin-permeabilized Calu-3 monolayer (see Methods). To isolate apical Cl⁻ conductance, the apical membrane Cl⁻ current (I_Cl) was measured after the apical to basolateral Cl⁻ gradient was established. An inward I_Cl is consistent with an absorptive Cl⁻ flow. (a) Under this condition, Cl⁻ conductance was continuously augmented by ATP (100 μM) added to the apical solution. (b) Even in the cells pretreated with PAR2AP (50 μM), the ATP-induced Cl⁻ conductance was not significantly affected. (c, d) Effects of PAR2 activation on ATP-induced K⁺ conductance across the basolateral membrane in a nystatin-permeabilized Calu-3 monolayer (see Methods). Basolateral membrane K⁺ conductance was estimated through basolateral membrane K⁺ current after establishment of an apical-to-basolateral K⁺ gradient. (c) K⁺ conductance was increased by additon of ATP (100 μM) to the apical solution. (d) Application of PAR2AP induced a transient increase in I_K. After exposure to PAR2AP (50 μM, basolateral) for 30 min, an ATP-induced increase in K⁺ conductance was attenuated. These experiments were performed under closed-circuit conditions.

In addition, fluo-3 signal measurement revealed that apically applied ATP (100 μM) elevated [Ca²⁺]_i in an oscillatory fashion (Figure 8a). The ATP-induced [Ca²⁺]_i changes were markedly attenuated by the presence of D609 (a PC-PLC inhibitor) or PMA (a PKC activator), and were also suppressed by the PAR2AP pretreatment (Figure 8b–d). Namely, Δ[Ca²⁺]_i was reduced from 1288.5±356.2 nM (n=9, Figure 8a) to 178.1±51.2 nM (n=6, P<0.05, Figure 8d) by the presence of PAR2AP. Further, the PAR2-mediated attenuation of [Ca²⁺]_i movement was reversed by the PKC inhibitor GFX (1 μM) up to 423.8±59.9 nM (n=6, P<0.05 vs without GFX, Figure 8e).

Figure 8.

(a–e) Effects of PARAP2 on extracellular ATP-induced Ca²⁺ mobilization in Calu-3 cells preloaded with fluo-3. Application of ATP (100 μM, apical) on the apical side increased [Ca²⁺]_i in a oscillatory fashion (a). The ATP-induced Ca²⁺ mobilization, which was significantly suppressed by D609 (100 μM, b) or PMA (1 μM, c), was also abolished by pretreatment with the PAR2-activating peptide (PAR2AP, 50 μM, basolateral, d). The PAR2-mediated attenuation was blunted by pretreatment with the PKC inhibitor GF109203X (GFX, 1 μM, e). D609, PMA, PAR2AP, and GF109203X were applied 30 min before addition of ATP.

Discussion

Thrombin (a PAR1, -3, -4 agonist) and trypsin (a PAR2, -4 agonist) are well-characterized enzymatic activators of PARs (Dery et al., 1998). We showed in human airway epithelial Calu-3 cells that trypsin, but not thrombin, applied to the basolateral face of the monolayer caused a transient change in ion transport that was mimicked by the application of PAR2AP, suggesting selective PAR2 expression. Our RT–PCR experiments to detect PAR2 mRNA verified this point in the present study. When trypsin and PAR2AP were used to determine concentration-dependent responses, apical application of these PAR2 agonists caused much smaller effects than basolateral application did. These observations indicate predominant localization of PAR2 on the basolateral membrane of Calu-3 cells, in accordance with native and cultured airway epithelial cells (Danahay et al., 2001; Kunzelmann et al., 2002). The most likely second messenger in the regulation of PAR2-mediated ion transport is cytosolic Ca²⁺, because PAR2-mediated I_eq changes were well correlated to those in [Ca²⁺]_i, as has been reported in normal human airway epithelial cells (Danahay et al., 2001). Calu-3 cells display the phenotype of CFTR-rich airway epithelium without any component of amiloride-sensitive Na⁺ absorption, and [Ca²⁺]_i elevation causes Cl⁻ secretion but neither HCO₃⁻ secretion nor Na⁺ absorption (Moon et al., 1997; Singh et al., 1997; Devor et al., 1999). This would render it conceivable that PAR2-stimulated ion transport in Calu-3 cells essentially comprises Cl⁻ secretion. This speculation was confirmed by the results that PAR2AP-induced responses were markedly suppressed by removal of Cl⁻ from the extracellular solutions and the presence of basolateral bumetanide, an inhibitor of Cl⁻ uptake across the basolateral membrane via the Na⁺–K⁺–2Cl⁻ cotransporter. Although Calu-3 cells almost exclusively express CFTR without Ca²⁺-dependent Cl⁻ channels (Haws et al., 1994), Ca²⁺-dependent CFTR-mediated Cl⁻ secretion does occur. Namely, activation of basolateral Ca²⁺-activated K⁺ channels provides a driving force for Cl⁻ efflux across the apical CFTR at rest (Moon et al., 1997). In Calu-3 cells, the majority of basolateral K⁺ conductance can be accounted for by the hIK-1 channel (an intermediate conductance Ca²⁺-activated K⁺ channel) because only a small cAMP-activated K⁺ conductance but a much larger hIK-1-dependent K⁺ conductance could be identified in the permeabilized monolayer (Devor et al., 1999; Cowley & Linsdell, 2002; Ito et al., 2004b; Szkotak et al., 2004). Preliminarily, we ascertained that PAR2-induced I_eq was significantly inhibited by the hIK-1 channel inhibitors charybdotoxin and clotrimazole (data not shown). Thus, it is most likely that PAR2-induced Cl⁻ secretion via resting CFTR is generated by Ca²⁺-dependent activation of the basolateral hIK-1 channel (see Figure 7b and d).

In most cellular systems, the key enzyme for the regulation of intracellular Ca²⁺ mobilization is G protein-coupled PLC. PLC is classified into PI-PLC and PC-PLC (Camina et al., 1999). PI-PLC stimulates the breakdown of phosphatidylinositol 4,5-biphosphate at the internal leaflet of the plasma membrane to form two second messengers, InsP₃ and DAG (Nakamura, 1996). InsP₃ triggers Ca²⁺ release from the endoplasmic reticulum, while DAG activates transient receptor potential channels, Ca²⁺ entry pathways (Kiselyov & Muallem, 1999). There are several lines of evidence that PAR-mediated Ca²⁺ mobilization is coupled to PI-PLC (Mule et al., 2002; Oshiro et al., 2002). Nevertheless, the results in Figure 4 revealed that the PAR2-mediated responses were unaffected by PI-PLC inhibitors, such as U73122 (K_i=4–10 μM) (Connor et al., 2001; Saeed et al., 2003; Rasheed & Saeed, 2004) or ET-18-OCH₃ (K_i=2–18 μM) (Powis et al., 1992; Daniel et al., 1995; Seebeck et al., 1998), and an InsP₃ inhibitor, Xest C (K_i=0.3–0.4 μM) (Gafni et al., 1997). However, recent studies have shed light on PC-PLC as an alternative pathway for production of DAG (Snetkov et al., 2001). In the present study, PAR2AP-induced Cl⁻ secretion and Ca²⁺ mobilization were markedly diminished by pretreatment with a specific PC-PLC inhibitor (D609), whose K_i on PC-PLC has been reported to be 5–20 μM (Tschaikowsky et al., 1994; Amtmann, 1996; Kengatharan et al., 1996; Nakahata et al., 2000). These results, thereby, suggest the possible coupling of PAR2 with PC-PLC on the basolateral membrane of Calu-3.

PAR-mediated activation of PC-PLC and resultant generation of DAG should activate PKC as well as mobilize Ca²⁺ (Nystedt et al., 1994). PKC has been reported to be involved in regulating PAR2-mediated Ca²⁺ mobilization in kidney and intestinal epithelial cell lines (Bohm et al., 1996). To investigate the effect of PKC on PAR2-mediated Cl⁻ secretion in Calu-3 cells, cells pretreated with PMA, a PKC activator, were exposed to PAR2AP, showing that PAR2 on Calu-3 cells seem likely to be under the regulation of PKC (see Figure 4e and f). From these observations, we predicted that PKC activation via PAR2 stimulation is involved in its auto-desensitization, and consequently we found it partially involved. To put it concretely, auto-desensitization during the continuous exposure to PAR2AP, however, was unaffected by a PKC inhibitor (Figure 5a and b), whereas auto-desensitization during the intermittent exposure to PAR2AP was mitigated by the PKC inhibition (see Figure 5d and e). In other words, PAR2-mediated PKC activation participates in extension of PAR2 auto-desensitization even after recovery from PKC-independent auto-desensitization (i.e., uncoupling from G-protein, or internalization into early endosomes) (Dery et al., 1998; Macfarlane et al., 2001).

PKC could be implicated in cross-desensitization between different receptors, supposing auto-desensitization of PAR2 involves PKC, which may act at the level of the receptor or at more distal steps in the signaling pathway (Bohm et al., 1996). Thus, we examined the effects of PAR2 activation on the anion secretion due to heterologous stimuli in Calu-3 cells. As shown in Figure 6, anion secretion generated by cAMP-related agents, such as isoproterenol, forskolin, and adenosine, were unaffected by the PAR2 activation through PAR2AP, suggesting that PAR2 activation does not influence signals via AC-coupled receptors (i.e., β₂-adrenergic receptor, A_2B adenosine receptor), the cAMP-PKA cascade, and cAMP-dependent channels (i.e., CFTR, cAMP-dependent K⁺ channels). Thapsigargin inhibits the Ca²⁺-ATPase that contributes to the Ca²⁺ uptake of intracellular Ca²⁺ into its store sites, leading to [Ca²⁺]_i elevation (Kim et al., 1998). Depletion of Ca²⁺ in the store sites due to thapsigargin also stimulates Ca²⁺ entry from the extracellular space (Takemoto et al., 1998). [Ca²⁺]_i elevation causes an opening of the basolateral hIK-1 channel, leading to Cl⁻ secretion, as described above. Thus, to determine whether Ca²⁺ mobilization itself or the hIK-1 channel was a target of PAR2-mediated desensitization, we examined the effects of PAR2 activation on the thapsigargin- and 1-EBIO (a hIK-1 opener)-induced anion secretion, but found no significant effect. Notwithstanding these observations, we found in this study that ATP- and ADPβS-induced anion secretion was desensitized by the pretreatment with PAR2AP. Previous studies to detect subtypes of P2Y receptors revealed that only P2Y₁R mRNA is expressed in Calu-3 cells (Communi et al., 1999), and extracellular ATP and ADPβS is well capable of stimulating this purinoceptor (Ralevic & Burnstock, 1998; Son et al., 2004). We have previously demonstrated that stimulation of P2Y₁R, which is coupled to both AC and PC-PLC, simultaneously causes CFTR activation and Ca²⁺ mobilization, generating Cl⁻ secretion (Son et al., 2004). The locus of the P2Y₁R-mediated signal transduction affected by PAR2 activation is not at the receptor levels but in the stream of the PC-PLC-mediated pathway, because, as shown in Figures 7 and 8, CFTR-mediated apical Cl⁻ conductance augmented by the addition of ATP was unchanged under PAR2-prestimulated conditions, while pretreatment with PAR2AP markedly attenuated ATP-mediated Ca²⁺ mobilization and elevation of basolateral K⁺ conductance that is predominantly mediated by a Ca²⁺-activated K⁺ channel hIK-1 in Calu-3 cells (Son et al., 2004). Further, the PAR2-mediated inhibition of ATP-induced Ca²⁺ mobilization was counteracted by the presence of a PKC inhibitor GFX. Therefore, we conclude that PC-PLC-mediated PKC activation through PAR2 causes a selective cross-desensitization of the P2Y₁R–PC-PLC pathway.

Of the PAR2-mediated auto-and cross-desensitizations, PKC activation via PC-PLC may act on G proteins, PC-PLC, or later steps in the signaling pathway. Nevertheless, it is more likely that G-proteins are not a principal target of PKC because of the previous findings that the G_qα-subunit, which is at least partially involved in PAR2- and P2Y₁-transduction (Ralevic & Burnstock, 1998; Macfarlane et al., 2001), is not phosphorylated by PMA (Lounsbury et al., 1993). Further, it has been reported that thapsigargin-induced Ca²⁺ mobilization, which acts on distal steps in the PLC pathway, is insensitive to PMA (Munshi et al., 1993), suggesting less involvement of PKC in the distal steps of PC-PLC. Add results shown in Figure 6c is also suggestive regarding this point. Alternatively, PC-PLC seems more likely to be a target for PAR2-mediated, PKC-dependent desensitization because both PC-PLC and PI-PLC are phosphorylated in response to activation of PKC by the PKC activator PMA or through PLC-coupled receptors, preventing interaction with G_i- and G_q-proteins (Camina et al., 1999). Considering the results obtained from the present study, we propose the hypothetical scheme of PAR2-mediated regulation of P2Y₁R shown in Figure 9.

Figure 9.

Possible mechanisms underlying PAR2-mediated desensitization related to PKC in Calu-3 cells. (+) and (−) indicate stimulatory and inhibitory effects, respectively. Both basolateral PAR2 and apical P2Y₁ purinoceptor (P2Y₁R) are coupled with PC-PLC, which hydrolyzes phosphatidylcholine at the plasma membrane. PC-PLC activity is inhibited by D609 and PMA. PAR2-mediated PC-PLC activation and the resultant formation of DAG are greatly responsible for Ca²⁺ mobilization, which activates the basolateral hIK-1 channel for Cl⁻ secretion, and PKC activation, which inhibits PC-PLC activity. The PAR2-mediated PKC activation leads to desensitization of PAR2 itself and P2Y₁R through PKC-to-PC-PLC negative feedback mechanisms. In this process, the adenylate cyclase (AC)-mediated pathway, which is also coupled with P2Y₁R, is intact.

In conclusion, PAR2 stimulation generates a brief response of Cl⁻ secretion through PC-PLC-mediated pathway, followed by not only auto-desensitization of PAR2 itself but also cross-desensitization of a PC-PLC-coupled purinoceptor (P2Y₁R). In both types of desensitization, PKC-to-PC-PLC negative feedback mechanisms appear to be involved. As a corollary of these results, the auto- and cross-desensitization of PAR2 signal transduction may be one of the important systems regulating airway anion secretion.

Abbreviations

[Ca²⁺]_i

cytosolic Ca²⁺ concentration

CFTR

cystic fibrosis transmembrane conductance regulator

DAG

diacylglycerol

1-EBIO

1-ethyl-2-benzimdazolinone

GFX

GF109203X

G_t

transepithelial conductance

hIK-1 channel

human intermediate conductance, inward-rectifying Ca²⁺-activated K⁺ channel

I_Cl

apical membrane Cl⁻ current

I_eq

equivalent short-circuit current

I_K

basolateral membrane K⁺ current

Ins P₃

inositol 1,4,5-triphosphate

NHBE

normal human bronchial epithelial cells

PAR2

protease-activated receptor 2

PAR2AP

PAR2-activating peptide

PC-PLC

phosphatidylcholine-phospholipase C

PD

transepithelial potential difference

PI-PLC

phosphatidylinositol-phospholipase C

PKC

protein kinase C

PSS

physiological saline solution

P2Y₁R

P2Y₁ purinoceptor

revPAR2AP

the reverse sequence peptide of PAR2-activating peptide

Xest C

xestospongin C