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Am J Physiol Cell Physiol 280: C1455-C1464, 2001;
0363-6143/01 $5.00
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Vol. 280, Issue 6, C1455-C1464, June 2001

Protease-activated receptor-2-mediated inhibition of ion transport in human bronchial epithelial cells

Henry Danahay1, Louise Withey1, Christopher T. Poll1, Stan F. J. van de Graaf2, and Robert J. Bridges2

1 Novartis Horsham Research Centre, Horsham, West Sussex RH12 5AB, United Kingdom; and 2 Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A cytoprotective role for protease-activated receptor-2 (PAR2) has been suggested in a number of systems including the airway, and to this end, we have studied the role that PARs play in the regulation of airway ion transport, using cultures of normal human bronchial epithelial cells. PAR2 activators, added to the basolateral membrane, caused a transient, Ca2+-dependent increase in short-circuit current (Isc), followed by a sustained inhibition of amiloride-sensitive Isc. These phases corresponded with a transient increase in intracellular Ca2+ concentration and then a transient increase, followed by decrease, in basolateral K+ permeability. After PAR2 activation and the addition of amiloride, the forskolin-stimulated increase in Isc was also attenuated. By contrast, PAR2 activators added to the apical surface of the epithelia or PAR1 activators added to both the apical and basolateral surfaces were without effect. PAR2 may, therefore, play a role in the airway, regulating Na+ absorption and anion secretion, processes that are central to the control of airway surface liquid volume and composition.

trypsin; epithelial sodium channel; human airway


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PROTEASE-ACTIVATED RECEPTORS (PARs) are a family of seven-transmembrane G protein-coupled receptors (7, 11). These receptors are activated by the proteolytic cleavage of a receptor-bound, NH2-terminal tethered ligand domain, which is then able to bind to the receptor and initiate signaling. To date, four PARs (PAR1-4) have been characterized (11). Thrombin activates PAR1, PAR3, and PAR4, whereas trypsin and mast cell tryptase activate PAR2 and possibly PAR4 (11). Synthetic peptides resembling the tethered ligand sequence of the PARs (with the exception of PAR3) can bind and activate the receptors and are useful tools for functional studies.

The functional relevance of PARs has been the subject of intense study because of their widespread systemic distribution and potential roles in numerous systems, including hemostasis (7), inflammation (5, 25), the regulation of smooth muscle tone (4, 20), and ion transport processes (1, 6, 24). PAR2 has been identified in a number of epithelial tissues and has been observed to have both protective and proinflammatory activities. PAR2 has been described as a "sentry for inflammation" due to its linkage to a variety of cytoprotective pathways (5). A protective role for PAR2 has been proposed in the pancreatic duct epithelium in which PAR2 activation induced a Cl- secretory response that would potentially "flush" bacterial toxins or microorganisms out of the system (24). A similar PAR2-mediated activation of Cl- secretion described in the M-1 kidney cortical collecting duct cell line might also represent a flushing/diluting mechanism (1). PAR2 activation has further been demonstrated to trigger glandular mucin secretion (15), another cytoprotective mechanism. However, with the exception of thrombin-mediated activation of PAR1 in the vasculature, the relevant endogenous activators of PAR2 are still largely the subject of speculation.

The regulation of ion transport processes in the airway epithelium is highly coordinated, and, dysregulation, as in cystic fibrosis, can be catastrophic to the normal functioning of the lung. The maintenance of a hydrated airway lining is essential for efficient mucociliary clearance (18), a primary defense mechanism for the removal of inhaled debris or microbes. The aim of the present study was to establish whether PAR2 could regulate ion transport processes in a differentiated culture of normal human bronchial epithelial cells (HBECs).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture

HBECs (Biowhittaker, UK) were cultured using a modification of the method described by Gray and colleagues (9). Cells were seeded into plastic T-75 flasks and grown in bronchial epithelial cell growth medium (BEGM; Biowhittaker) supplemented with bovine pituitary extract (52 µg/ml), hydrocortisone (0.5 µg/ml), human recombinant epidermal growth factor (0.5 µg/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), retinoic acid (0.1 µg/ml), triiodothyronine (6.5 µg/ml), gentamycin (50 µg/ml), and amphotericin B (50 µg/ml). Medium was changed every 48 h until cells were 90% confluent. Cells were then passaged and seeded onto polycarbonate Snapwell inserts (Costar, UK) or 25-mm glass coverslips (Fisher Scientific) in differentiation media containing 50% DMEM in BGEM with the same supplements as above but without triiodothyronine and a final retinoic acid concentration of 50 nM (all-trans-retinoic acid). Cells were maintained submerged for the first 7 days in culture, after which time they were exposed to an apical air interface for the remainder of the culture period. Cells on coverslips were maintained submerged during the entire culture period. Cells on inserts were used between days 14 and 21 after establishment of the apical air interface, and cells on coverslips were used between days 7 and 14 after seeding. At all stages of culture, cells were maintained at 37°C in 5% CO2 in an air incubator. All studies were performed on cultures from three individual donors.

Short-Circuit Current Measurements

Snapwell inserts were mounted in Costar vertical diffusion chambers and were bathed with continuously gassed Ringer solution (5% CO2 in O2; pH 7.4) maintained at 37°C containing (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose. The solution osmolarity was always between 280 and 300 mosmol/kgH2O for all physiological salt solutions used. Cells were then voltage clamped to 0 mV (EVC4000, World Precision Instruments). Transepithelial resistance (RT) was measured by either applying a 2-mV pulse at 60 s intervals or by measuring the potential difference under open-circuit conditions and calculating RT by Ohm's law. Data were recorded using a PowerLab workstation (ADInstruments, UK). For Cl--free studies, sodium chloride was replaced by equimolar sodium gluconate. In studies to evaluate the basolateral membrane K+ current, the apical membrane was permeabilized with nystatin (180 µg/ml; apical side only) with the apical solution containing potassium gluconate (120 mM) in place of sodium chloride and the basolateral solution containing sodium gluconate, again in the place of sodium chloride. Nystatin was added to the apical membrane 5-10 min after voltage clamping and was present throughout the experiment. In all gluconate-containing solutions, the Ca2+ concentration was increased to 4 mM to compensate for the Ca2+-chelating property of gluconate (3).

Intracellular Ca2+ Concentration Measurements

HBECs on 25-mm glass coverslips were incubated at room temperature with 5 µM fura 2-AM in a bath solution containing (in mM) 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.4 using NaOH) for 45 min. Cells were washed with a fura 2-AM-free bathing solution and incubated at room temperature for 45 min to allow for the further hydrolysis of intracellular fura 2-AM to fura 2. Coverslips were then mounted in a 0.5-ml perfusion chamber, cells facing upward, on a heated stage fixed at 37°C. Fluorescence was measured with an inverted microscope (Nikon Diaphot 300) equipped with a ×40 oil-immersion objective. Fluorescence was monitored at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Images were acquired every 2 s using an intensified charge-coupled device camera (C4742-95; 640 × 512 pixels resolution; Hamamatsu Photonics, Bridgewater, NJ). The Ca2+ concentration was calculated by in vivo calibration using the following formula
[Ca<SUP>2+</SUP>]<SUB>i</SUB><IT>=K</IT><SUB>d</SUB><IT>×</IT>(R<IT>−</IT>R<SUB>min</SUB>)(R<SUB>max</SUB><IT>−</IT>R)<IT>×&bgr;</IT>
where the dissociation constant (Kd) = 224 nM; R = ratio of fluorescence with excitation at 340 and 380 nm, respectively; Rmin and Rmax = fluorescence ratio with low (10 mM) EGTA and (10 µM) ionomycin and high Ca2+ (2 mM CaCl2 and 10 µM ionomycin); and beta  = ratio of fluorescence at 380 nm with low and high Ca2+ (10).

Compound Additions

The effects of PAR activators were studied on the spontaneous basal short-circuit current (Isc) established by the HBECs and on the development of a forskolin-dependent current following treatment with the epithelial Na+ channel blocker, amiloride. To investigate potential mechanisms of any PAR activity on these currents, experiments were also performed under Cl--free conditions and after apical permeabilization with nystatin to monitor the basolateral K+ permeability (see above). The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), as the cell-permeant acetoxymethyl ester, was used to examine the Ca2+ dependence of any PAR-mediated changes in ion transport. Effects of the PAR activator trypsin on intracellular Ca2+ concentration ([Ca2+]i) were further studied using the Ca2+-dependent fluorescent dye fura 2. Porcine trypsin and human thrombin were freshly prepared at a 1,000× stock concentration in Ringer solution and stored on ice. A selective peptide agonist of PAR1 (TFRIFD-NH2), a mixed PAR1/2 peptide (SFLLRN-NH2) (16), a selective PAR2 peptide (SLIGRL-NH2), and PAR2 peptide control (LSIGRL-NH2) (13) were all prepared at 100× stock concentrations in Ringer solution. Amiloride was prepared in a 1,000× stock concentration in water and added to the apical surface to achieve a final concentration of 10 µM. Forskolin was dissolved in DMSO and added to both sides of the Ussing chambers at a final concentration of 2 µM such that the final concentration of DMSO did not exceed 0.1% vol/vol.

RT-PCR and Cloning of PAR2 and Putative Endogenous PAR Activators

Unless otherwise stated, all reagents were used according to the manufacturer's instructions. Briefly, mRNA was extracted from HBEC cultures using a QIAGEN RNeasy Mini kit. The expression of mRNA transcripts for PAR2, human airway trypsin-like enzyme (HAT) and trypsinogen, was investigated using RT-PCR (Promega Access RT-PCR System). Samples were run in a Biometra T3 Thermocycler for 30 cycles using primers designed from GenBank sequences (Primer Express, PE Biosystems). Primers were synthesized by Sigma Genosys: 5'-cctgcagtggcaccatcc-3' (PAR2 forward), 5'-cagggagatgccaatggc-3' (PAR2 reverse), 5'-cctggcagtcaccatagctct-3' (HAT forward), 5'-agtcttcttgtactagtggg-3' (HAT reverse), 5'-acaagtcccgcatccaggt-3' (trypsinogen forward), and 5'-tggtgtccttaatccagtcc-3' (trypsinogen reverse). A further hot-start PCR was carried out on the RT-PCR products for HAT and trypsinogen to increase specificity and selectivity. Each reaction contained 5 µl of buffer (PCR reaction buffer containing MgCl2; Boehringer Mannheim), 1 µl of dNTP mix (Promega), 1 µl (300 µg) of forward primer, 1 µl (300 µg) of reverse primer, 1 µl of RT-PCR product (diluted 1/20), and 40 µl of diethyl pyrocarbonate-treated water. One microliter of Taq DNA polymerase (Boehringer Mannheim; 5 U/µl) was added to the tubes after the initial denaturation at 94°C while the reaction paused at 80°C. The hot-start PCR was run in a Biometra T3 Thermocycler (30 cycles of 60°C, 45 s right-arrow 72°C, 3 min right-arrow 95°C, 45 s, followed by one cycle at 72°C for 10 min). The additional primers used in hot-start PCR were designed as previously described and synthesized by Sigma Genosys: 5'-ccctcctcagcctcagtgc-3' (HAT), 5'-ccttggtgtagactccaggcc-3' (trypsinogen). All final products were electrophoresed on 2% agarose gels in 1× Tris-acetate-EDTA buffer containing 50 µg of ethidium bromide.

HAT and trypsinogen transcripts were sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems). Briefly, the bands from the appropriate reactions were excised from the gels and the DNA was extracted using a QIA quick gel extraction kit (Qiagen). Sequencing reactions were prepared and run in a Biometra T3 Thermocycler (30 cycles of 96°C 30 s right-arrow 50°C, 15 s right-arrow 60°C 4 min). After ethanol precipitation, the final product was resuspended in 25 µl of template suppression reagent (ABI Prism DNA sequencing kit) and sequenced on an ABI Prism 310 sequencer, and identity was confirmed by comparison with GenBank sequences. PAR2 was cloned using the TOPO cloning system and TOP10 One Shot Chemical transformation kit (Invitrogen). Plasmids were analyzed by restriction analysis, isolated using the QIAGEN Plasmid Mini kit (Qiagen), and sequenced as described above.

Expression of Results and Statistical Analysis

Results are expressed as absolute changes in Isc (means ± SE). Measurements were taken either as peak changes or once responses had plateaued and were stable. Control inserts were run alongside all experiments for paired comparisons to be made owing to the potential day-to-day and interbatch variability of the Isc. A Student's t-test was used to compare between groups with statistical significance assumed when P < 0.05.

Reagents

HBECs obtained from postmortem specimens were purchased from Biowhittaker, as were all media. All other cell culture reagents were purchased from Life Technologies (UK). PAR peptide agonists were purchased from Sigma Genosys (UK). Fura 2-AM was purchased from Molecular Probes. All other reagents were purchased from Sigma (UK).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HBEC Characteristics

The culture methods employed produced a multilayered bronchial epithelial tissue that had differentiated to the extent that ciliated and goblet cells were identifiable. Goblet cells typically accounted for 20-25% of the total number of cells (data not shown). When voltage clamped to 0 mV, the cells displayed an Isc of 9.1 ± 0.3 µA/cm2 (n = 90) and an RT of 972 ± 34 Omega /cm2 (n = 90). Amiloride added to the apical solution caused a concentration-dependent decrease in Isc with an IC50 of 220 ± 10 nM (n = 6) and a maximal inhibition of baseline Isc of 82.1 ± 2.8% (n = 5). The amiloride-sensitive Isc was used as a measure of electrogenic Na+ transport.

Trypsin and Thrombin on Basal Isc

Trypsin (1 µM), when added to the basolateral medium, caused a transient increase in Isc of 12.5 ± 1.5 µA/cm2 above baseline that was followed by a sustained reduction in Isc of 4.4 ± 0.5 µA/cm2 below the initial baseline and a 38.0 ± 3.3% increase in RT (n = 4; Fig. 1). The subsequent addition of amiloride (10 µM) to the apical medium reduced the Isc by a further 3.6 ± 0.3 µA/cm2, compared with a decrease of 5.8 ± 0.2 µA/cm2 in the paired control cells (P < 0.05; n = 6). The decrease in Isc below basal levels observed with the combination of trypsin and amiloride of 8.0 ± 0.4 µA/cm2 was significantly greater than the decrease of 5.8 ± 0.2 µA/cm2 seen with amiloride alone (P < 0.05), indicating that trypsin also inhibits a proportion of the amiloride-insensitive current.


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  		Fig. 1.   Current traces illustrating the effects of porcine trypsin (1 µM) and human thrombin (5 U/ml) on the basal short-circuit current (Isc) and subsequent amiloride-sensitive and forskolin-stimulated Isc. A: control filter illustrating the responses to amiloride and forskolin. B: trypsin, added to the basolateral membrane, caused a transient increase in Isc, followed by sustained decrease and subsequent reduced amiloride-sensitive current. The combined reduction in Isc with trypsin and amiloride was greater than that seen with amiloride alone (A). Trypsin also attenuated the forskolin-stimulated increase in Isc. C: apical administration of trypsin (1 µM) was without significant effect. D: likewise, human thrombin (5 U/ml apical and basolateral) had only a small transient effect on Isc. AM, amiloride (10 µM apical); FSK, forskolin (2 µM apical and basolateral). Vertical deflection (A and B) represents the current responses to 2-mV pulses. Monolayers in C and D were not pulsed.

The addition of forskolin in the presence of amiloride caused an increase in Isc of 12.5 ± 0.4 µA/cm2 (n = 6) in control cells that was significantly attenuated in the trypsin-treated cells to 4.0 ± 0.6 µA/cm2 (n = 4; P < 0.01). Heat-inactivated trypsin added to the basolateral surface was without effect on any of the currents studied (data not shown). Likewise, there was no effect of trypsin when added apically or to thrombin (5 U/ml) added to either the apical or basolateral solution on the Isc (Fig. 1, C and D).

Dose Dependence of Trypsin and PAR Peptide Effects

The effects of trypsin on the ion transport characteristics described above were concentration dependent and observed over a concentration range of 100-1,000 nM (Fig. 2). These phenomena were also observed with the PAR2-activating peptide, SLIGRL-NH2 (10-100 µM; Figs. 2 and 3), and the mixed PAR1/2-activating peptide, SFLLRN-NH2 (15 and 100 µM), added to the basolateral membrane (Fig. 3). SLIGRL-NH2 (100 µM) caused a transient increase in Isc of 12.5 ± 0.8 µA/cm2, followed by a decrease below the initial basal current of 2.7 ± 0.5 µA/cm2 (n = 4). The residual amiloride-sensitive Isc of 1.7 ± 0.2 µA/cm2 was also significantly reduced when compared with the paired control amiloride-sensitive Isc of 2.9 ± 0.1 µA/cm2 (P < 0.05). The subsequent forskolin-stimulated current increase of 3.3 ± 0.3 µA/cm2 in the SLIGRL-NH2-stimulated group was also significantly reduced when compared with the paired control increase of 8.4 ± 0.3 µA/cm2 (P < 0.05). A scrambled control PAR2 peptide (LSIGRL-NH2, 100 µM) was without effect on the Isc. Basolateral SFLLRN-NH2 (100 µM), the mixed PAR1/PAR2 peptide, induced a transient increase in current of 3.5 ± 0.1 µA/cm2 followed by a decrease of 6.0 ± 0.3 µA/cm2 below the basal current (n = 4). The amiloride-sensitive current was reduced from 10.4 ± 0.7 µA/cm2 in the paired control group to 4.4 ± 0.3 µA/cm2 in the SFLLRN-NH2-treated cells (P < 0.05). SFLLRN-NH2 also reduced the forskolin-stimulated increase in Isc from 11.9 ± 0.8 µA/cm2 in paired control cells to 7.0 ± 0.4 µA/cm2 (P < 0.05). The selective PAR1 peptide, TFRIFD-NH2 (100 µM), was without effect (Fig. 3C; n = 4).


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  		Fig. 2.   Dose-response effects of human bronchial epithelial cells (HBECs) to trypsin and SLIGRL-NH2 (both basolateral) in terms of peak transient increases in Isc (A and B), reduction in Isc below initial control level (C and D), residual amiloride-sensitive current (E and F), and the forskolin-stimulated current (G and H). Data for plots C-H measured at response plateau. Data shown represent absolute changes in Isc expressed as means ± SE (n = 4-6).



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  		Fig. 3.   Current traces illustrating the effects of basolateral SLIGRL-NH2 (B; 30 µM), SFLLRN-NH2 (C; 15 µM), and TFRIFD-NH2 (D; 100 µM) on basal, residual amiloride-sensitive and forskolin-dependent currents. A: a typical control trace.

Potential Mechanisms of PAR2-Mediated Effects on Ion Transport

Ca2+ dependence. PAR2-mediated effects in other cell types have been shown to be mediated by an increase in [Ca2+]i. This signaling mechanism was investigated in HBECs using the fluorescent Ca2+-sensitive dye fura 2 and by buffering intracellular Ca2+ using BAPTA. Loading the cells with BAPTA (50 µM, 60 min) had no effect on the basal characteristics of the epithelium. Under these conditions, the initial transient increase in Isc observed following trypsin (1 µM) was reduced from 23.5 ± 2.9 µA/cm2 to 1.1 ± 0.4 µA/cm2 (P < 0.05, n = 4; Fig. 4). There was, however, no effect of BAPTA loading on the decrease in Isc, with trypsin causing reductions in Isc of 5.6 ± 0.4 µA/cm2 and 5.2 ± 0.1 µA/cm2 in the absence and presence of loaded BAPTA, respectively (P > 0.05, n = 4). A similar phenomenology was observed with SLIGRL-NH2 after BAPTA loading with the transient response to basolateral SLIGRL-NH2 (30 µM) being reduced from 5.5 ± 1.4 µA/cm2 to 2.7 ± 0.3 µA/cm2 (P < 0.05, n = 4), while the inhibitory phase remained unaffected (control 3.9 ± 0.8 µA/cm2 vs. BAPTA loaded 2.8 ± 0.2 µA/cm2, P > 0.05). The trypsin-mediated effects on [Ca2+]i were determined using fura 2. HBECs, grown on coverslips, were loaded with fura 2, and fluorescence was measured while cells were bathed in Ca2+-containing solution. The [Ca2+]i was calculated as described in METHODS. The basal Ca2+ concentration was 43 ± 13 nM. Addition of trypsin (1 µM) caused a rapid increase in [Ca2+]i to 321 ± 51 nM. After 1.5-4 min, [Ca2+]i returned to a level that was not significantly different from the basal level (41 ± 24 nM compared with 43 ± 13 nM, P > 0.05, n = 28 cells, 5 experiments; Fig. 5).


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  		Fig. 4.   Current traces illustrating the effects of vehicle (A and C; 0.1% DMSO) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; B and D; 50 µM, 60 min) on the effects caused by trypsin (A and B; 1 µM) and SLIGRL-NH2 (C and D; 30 µM) on the basal HBEC current.



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  		Fig. 5.   Ca2+ trace illustrating the rise in intracellular Ca2+ concentration ([Ca2+]i) induced by the addition of trypsin (1 µM) to HBECs cultured on glass coverslips. HBECs were loaded with fura 2-AM (5 µM) for 45 min at room temperature before stimulating at 37°C. Fluorescence was monitored at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm.

Cl--free conditions. Cl--free conditions were used to establish whether the PAR2-mediated effects on Isc were dependent on Cl- transport. In the absence of Cl-, trypsin (1 µM) caused an increase in Isc of 9.2 ± 0.4 µA/cm2 followed by a sustained decrease of 3.5 ± 0.1 µA/cm2 (n = 6) below the initial basal current (Fig. 6). Under Cl--free conditions, SLIGRL-NH2 (100 µM) caused an increase in Isc of 8.2 ± 0.4 µA/cm2 and a sustained decrease in current of 2.0 ± 0.2 µA/cm2 (n = 4).


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  		Fig. 6.   Current traces illustrating the effects of basolateral trypsin (A; 1 µM) and SLIGRL-NH2 (B; 30 µM) in Cl--free media.

Basolateral membrane K+ currents. These experiments were used to establish whether K+ channels on the basolateral membrane played a role in the PAR2-mediated effects on Isc. Permeabilization of the apical membrane with nystatin caused an immediate increase in K+ currents (IK) under the established apical-to-basolateral K+ gradient (Fig. 7). The addition of either trypsin (1 µM) or SLIGRL-NH2 (30 µM) induced a transient increase in Isc of 246.1 ± 8.0 µA/cm2 (n = 6) and 39.5 ± 5.6 µA/cm2 (n = 4) above the nystatin-induced baseline, respectively. The trypsin- and SLIGRL-NH2-induced transient increases then decreased to new baselines of 21.4 ± 0.8 µA/cm2 and 14.8 ± 3.6 µA/cm2 below the nystatin-permeabilized baseline, respectively.


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  		Fig. 7.   Current traces illustrating the effects of basolateral trypsin (A; 1 µM) and SLIGRL-NH2 (B; 30 µM) on Isc under conditions of an imposed apical-to-basolateral K+ gradient following permeabilization of the apical membrane with nystatin (180 µg/ml).

Expression of PAR2 and Putative Endogenous PAR2 Activators in the HBEC Model

RT-PCR of isolated RNA from the HBECs used in these studies revealed the expression of PAR2 (Fig. 8). The identities of endogenous activators of PAR2 in the airway are unknown and the subject of speculation. Consequently, we examined the expression of two putative endogenous PAR2 activators in the cell culture system employed for the Isc studies. RT-PCR and subsequent sequencing revealed the expression of trypsinogen and HAT in the cultured cells (Fig. 8).


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  		Fig. 8.   Products of RT-PCR reactions of RNA isolated from differentiated HBECs using gene-specific primer pairs targeted to PAR2 (lane 1), human trypsinogen (lane 2), and human airway trypsin-like protease (lane 3). Sequencing confirmed their identity.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PAR2 Activation From the Basolateral Surface

The bronchial epithelial cells used in this study displayed a spontaneous Isc that was 60-80% amiloride sensitive, as previously described for human airway epithelial cultures (8, 9, 29). Robust effects on ion transport were only observed with PAR2 activators (trypsin and PAR2-activating peptides) and not with PARs 1, 3, and 4 (thrombin or TFRIFD-NH2). The responses to trypsin and the PAR2-activating peptides were the same, suggesting that they share a common target, and it is unlikely that the trypsin-induced effect is via cleavage of a protein other than PAR2. PAR2 activation caused a transient increase in Isc followed by an inhibition of amiloride-sensitive Isc. The observation that the combination of PAR2 activator and amiloride caused a greater reduction in basal current than amiloride alone suggests that PAR2 activation also attenuates some of the amiloride-insensitive current. The subsequent forskolin-stimulated increase in Isc was also attenuated. Furthermore, PAR2-induced effects were only observed upon basolateral addition of the activators. It is interesting to note that in tissue sections of human airways, PAR2 was not exclusively localized to the basolateral membrane by immunohistochemistry (4). However, PAR2 was resolved exclusively to the basolateral membrane both functionally and histologically in pancreatic duct epithelium (24). The basolateral dependency for the PAR2 responses in this study is likely to represent receptor localization to the basolateral surface and not a differential membrane coupling to second messenger systems. Studies of P2Y2 agonist effects on ion transport in a variety of airway epithelia have described a similar phenomenology to that observed here, irrespective of apical or basolateral administration (8, 14, 22, 23). Like PAR2, P2Y2 receptors mediate their effects via an increase in intracellular Ca2+ as part of the signal transduction cascade in human airway epithelium (26). Therefore, the appropriate signaling apparatus, with respect to Ca2+ mobilization as well as Ca2+-activated ion channels, is located in both the apical and basolateral membranes. However, PAR2 agonists failed to have any detectable effects when added to the apical side, suggesting a lack of apically located receptors in the cultured cells.

PAR2-Induced Transient Increase in Isc

Trypsin and the PAR2-activating peptides (SLIGRL-NH2 and SFLLRN-NH2) caused a transient increase in basal Isc. In the majority of tissues, PAR2 couples to phospholipase Cbeta -forming D-myo-inositol 1,4,5-trisphosphate, which leads to Ca2+ mobilization from intracellular stores (7). The acute effect of PAR2 activation in this system appeared to be Ca2+ dependent, because measurement of intracellular Ca2+, using fura 2, demonstrated a transient increase in [Ca2+]i, and loading of the cells with the Ca2+ chelator, BAPTA, attenuated the Isc response (Fig. 4). The transient increase in Isc caused by PAR2 activation was also independent of the presence of Cl- in the system, as a qualitatively similar response to PAR2 activation was observed in the absence of Cl-. However, an effect on Cl- transport cannot be ruled out from these studies. The transient increase in Isc was observed in parallel with a transient trypsin-/PAR2 peptide-induced opening of a basolateral K+ conductance when measured in apically permeabilized cells under a K+ gradient. The opening of a basolateral K+ channel would be expected to hyperpolarize the cell and increase the driving force for Na+ entry via the epithelial Na+ channel. Devor and Pilewski (8) described a similar transient increase in Isc in cultured HBECs induced by a variety of Ca2+-activating agonists, including UTP. The increase in Isc was attributed to the opening of basolateral membrane Ca2+-dependent K+ channels that enhanced Na+ absorption (8). However, an additional stimulation of Cl- secretion cannot be ruled out because the change in membrane potential would also favor Cl- exit from the cell, an effect observed after P2Y2 stimulation in human distal lung alveolar cells (27) and in rabbit tracheal epithelium (14). Furthermore, PAR2-activated Cl- secretion that was attributable to the opening of a basolateral K+ channel has been previously reported by Nguyen and colleagues (24) in pancreatic duct epithelium. A PAR2-induced Cl- secretory current has also been described in M-1 cells (1) in which the opening of a Ca2+-activated Cl- channel was also observed, although an effect on K+ conductance could not be ruled out.

PAR2-Induced Sustained Decrease in Isc

The sustained decrease in Isc observed after the resolution of the transient increase in current was apparent with both trypsin and PAR2 peptide activation. The sustained decrease in Isc was observed in Cl--free solutions and was associated with a phase of decreased K+ conductance in the nystatin studies. A similar phenomena has been described with a variety of Ca2+-mobilizing stimuli, including P2Y2 agonists (8, 13, 22, 27). These observations would suggest that closure of the basolateral K+ conductance reduces the driving force for Na+ entry as well as any basal anion secretion. This is supported by the observation that the residual amiloride-sensitive current is significantly reduced. A reduction in basolateral K+ conductance would also account for the greater reduction in Isc observed with the combination of PAR2 activation and amiloride in this study when compared with amiloride alone. The addition of amiloride will hyperpolarize the cells and therefore increase the driving force for anion secretion. With a reduced K+ conductance of the basolateral membrane induced by PAR2 activation, the amiloride-induced hyperpolarization will be reduced, and the driving force for anion exit decreased. The lack of effect of BAPTA against this response suggests that a second messenger other than Ca2+ is responsible for the bulk of the inhibitory effect on the amiloride-sensitive and insensitive currents, a view supported by the work of Mason et al. (22), Devor and Pilewski (8), and Inglis et al. (13). In the kidney, the inhibitory effects of Ca2+-dependent agonists are believed to involve the activation of protein kinase C (PKC) (19, 21), although a variety of PKC inhibitors has been shown to be without effect on the UTP-induced inhibitory phase of Isc in cultured HBECs (8) and in porcine tracheal epithelium (13). It is, however, of note that Inglis and colleagues (13) did attenuate this response with staurosporine, suggesting that an as yet unidentified protein kinase may be involved. The inhibitory effect on Na+ transport by PAR2 activation was not observed in the studies of Bertog et al. (1) using M-1 cells despite the observed increases in intracellular Ca2+. This difference between the HBECs and M-1 cells may reflect differences in the K+ channels involved in Na+ transport across these epithelia.

PAR2-Induced Attenuation of Forskolin-Stimulated Isc

The inhibition of the forskolin-stimulated increase in Isc by PAR2 activation could also be explained in terms of an effect on basolateral K+ conductance. Forskolin is expected to activate both cystic fibrosis transmembrane conductance regulator and a basolateral K<UP><SUB>cAMP</SUB><SUP>+</SUP></UP> conductance to cause anion secretion. Inhibition of this K+ conductance would attenuate the driving force for anion efflux. It is likely, therefore, that PAR2 activation results in effects on at least two classes of K+ channel, namely the K+ conductance that supports Na+ absorption and the K+ conductance activated by forskolin (KcAMP). We have previously demonstrated a similar inhibitory effect of PAR2 activation on a forskolin-stimulated Cl- secretory current in T84 cells that was attributed to a large increase in basolateral resistance determined by impedance analysis (6).

The location of the PAR2 receptor to the basolateral surface implies that the endogenous ligand (protease) is either released in an autocrine manner by the epithelial cells or by an infiltrating inflammatory cell, or alternatively, the protease is perhaps a product of pathogens colonizing the airway mucosa. The most obvious source of a tryptic protease would be the mast cell, which is able to secrete mast cell tryptase, a recognized activator of PAR2 (7). Mast cell tryptase was not used in these studies because of the quantities that would have been required to achieve the working concentration in the Ussing chamber. It has also been noted that the cleavage site for human PAR2 (KGR/SLI) bears similarity to the activation site of complement factor C1s (KQR/IIG), raising the potential for complement activation to produce an activator of PAR2 (12). Cocks et al. (4) identified trypsinogen in human bronchial biopsies by immunohistochemistry, and we have confirmed the presence of mRNA for this trypsin precursor in our HBEC model. However, it remains to be determined whether an endogenous kinase (e.g., enterokinase) exists within the lung that would convert inactive trypsinogen to active trypsin. We have also identified the expression of HAT, a tryptic enzyme (28) that could potentially cleave and activate PAR2 in an autocrine manner. This enzyme has previously been identified in submucosal glands and in the sputum/bronchial lavage samples of patients suffering from chronic airway diseases (30). To our knowledge, the studies shown in Fig. 8 are the first to demonstrate the expression of HAT in HBECs. The presence of HAT in sputum and bronchial lavage samples would, however, localize the enzyme to the apical surface of the epithelium, and it remains to be shown whether it is also secreted to the basolateral side where it could activate PAR2.

This is the first description of a role for PAR2 in the regulation of ion transport processes in the airway epithelium and represents a putative mechanism for the endogenous control of basal Na+ reabsorption and both basal and stimulated anion secretion. Under basal conditions, Na+ absorption represents the major ion transport process (2) and plays a central role in the regulation of airway surface liquid (ASL) volume (17). It is important to note that Cl- secretion has not been detected under basal, unstimulated conditions in the human airway (2). The PAR2-mediated inhibition of basal amiloride-sensitive Isc, if physiologically relevant, would therefore be predicted to increase the ASL volume and potentially improve mucociliary clearance (17, 18). The physiological relevance of the PAR2-mediated attenuation of the basal amiloride-insensitive Isc and cAMP-stimulated Cl- secretion is unknown and may simply reflect a consequence of K+-channel inhibition to achieve the blockade of Na+ absorption. In summary, a PAR2-mediated decrease in Na+ absorption in the airway epithelium adds further evidence to a role for PAR2 as a first line of defense in epithelia. Further studies will be required to determine whether this mechanism is of functional relevance in vivo.


    FOOTNOTES

Address for reprint requests and other correspondence: H. Danahay, Novartis Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex RH12 5AB, United Kingdom (E-mail: henry.danahay{at}pharma.novartis.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 July 2000; accepted in final form 20 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 280(6):C1455-C1464
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