Protease-activated receptor-1 stimulates
Ca2+-dependent Cl
secretion in human
intestinal epithelial cells
M. C.
Buresi,
E.
Schleihauf,
N.
Vergnolle,
A.
Buret,
J. L.
Wallace,
M. D.
Hollenberg, and
W. K.
MacNaughton
Mucosal Inflammation Research Group, University of Calgary,
Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
The thrombin receptor, protease-activated receptor-1
(PAR-1), has wide tissue distribution and is involved
in many physiological functions. Because thrombin is in the intestinal
lumen and mucosa during inflammation, we sought to determine PAR-1
expression and function in human intestinal epithelial cells. RT-PCR
showed PAR-1 mRNA expression in SCBN cells, a nontransformed duodenal
epithelial cell line. Confluent SCBN monolayers mounted in Ussing
chambers responded to PAR-1 activation with a
Cl
-dependent increase in short-circuit current. The
secretory effect was blocked by BaCl2 and the
Ca2+-ATPase inhibitor thapsigargin, but not by the L-type
Ca2+ channel blocker verapamil or DIDS, the nonselective
inhibitor of Ca2+-dependent Cl
transport.
Responses to thrombin and PAR-1-activating peptides exhibited auto- and
crossdesensitization. Fura 2-loaded SCBN cells had increased
fluorescence after PAR-1 activation, indicating increased intracellular
Ca2+. RT-PCR showed that SCBN cells expressed mRNA for the
cystic fibrosis transmembrane conductance regulator (CFTR) and
hypotonicity-activated Cl
channel-2 but not for the
Ca2+-dependent Cl
channel-1. PAR-1 activation
failed to increase intracellular cAMP, suggesting that the CFTR channel
is not involved in the Cl
secretory response. Our data
demonstrate that PAR-1 is expressed on human intestinal epithelial
cells and regulates a novel Ca2+-dependent Cl
secretory pathway. This may be of clinical significance in inflammatory intestinal diseases with elevated thrombin levels.
thrombin; epithelium; ion transport; serine endopeptidases; chloride channels
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INTRODUCTION |
PROTEASE-ACTIVATED
RECEPTORS (PARs) are a unique class of G protein-coupled
receptors activated by serine proteases that cleave specific regions of
the extracellular NH2 terminus of the molecule to reveal a
new NH2 terminus that acts as a "tethered ligand." The
tethered ligand, by binding to other extracellular domains on the PAR
molecule, stimulates G protein-dependent signaling (34,
35). Four PARs have been cloned to date (17, 18, 24, 34,
38), and others have been predicted based on pharmacological structure-activity relationships (26, 28, 33). PAR-1, -3, and -4 are activated by the coagulation factor thrombin, whereas PAR-2
is activated by trypsin and possibly by mast cell tryptase (5,
21, 23). PAR-1 was the first PAR cloned and is the prototypical
thrombin receptor. It is found in a wide variety of cell types,
including platelets, endothelial cells, fibroblasts, monocytes, T cell
lines, osteoblast-like cells, smooth muscle cells, neurons, and glial
cells, and in certain tumor cell lines (7).
Thrombin has long been known to be involved in inflammation and has
been implicated in the pathogenesis of inflammatory bowel disease.
Thrombin and PAR-1 have critical proinflammatory effects such as
platelet aggregation, vasodilatation and vasoconstriction, increased
vascular permeability, and granulocyte chemotaxis (8). Patients with Crohn's disease show various coagulation abnormalities, and intestinal vascular injury has been proposed as a major pathogenic factor (30, 36). Chronic inflammatory bowel disease,
especially ulcerative colitis, is associated with a thrombotic tendency
(12). Thus thrombin would be in a position to affect
epithelial function in the inflamed gut.
The epithelium plays a central role in host defense. The ability of
epithelial cells in the crypt region to secrete chloride ions and water
prevents or slows the translocation of bacteria, bacterial products,
and antigens from the intestinal lumen to the mucosa (4).
A loss of basal secretion or of the ability to respond to secretagogues
renders the organism susceptible to infection. Conversely,
hyperresponsiveness to secretagogues tips the absorption and secretion
balance in the other direction, resulting in diarrhea that could lead
to excessive electrolyte and water loss. Electrolyte-transporting
epithelial cells from nonintestinal tissues, including renal
proximal tubule cells (13), have been shown to express
PAR-1. However, intestinal epithelial expression of PAR-1 has not been
demonstrated nor has thrombin been linked to intestinal epithelial cell
function or dysfunction.
In this study, we tested the hypothesis that PAR-1 is expressed on
intestinal epithelial cells and can modulate epithelial secretion of
chloride and, hence, water. To do this, we assessed the expression and
function of PAR-1 in a nontransformed, chloride-secreting human crypt
cell line. Furthermore, because chloride secretion by intestinal
epithelial cells can occur through calcium- or adenylate cyclase-dependent pathways, we conducted further experiments to determine which of these pathways is involved in PAR-1-induced chloride transport.
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MATERIALS AND METHODS |
SCBN cells.
SCBN is a nontransformed, chloride-secreting, duodenal epithelial crypt
cell line (25). SCBN cells were grown according to
previously published methods (25, 29). Briefly, cells of passages 23-33 were grown to confluence (~5 days) in
either 75-cm2 flasks or on Snapwell semipermeable supports
(Corning, Corning, NY). Cells in Snapwell supports were fed every day
with DMEM supplemented with 5% fetal bovine serum,
L-glutamine, streptomycin, and tylosin. Cells in flasks
were fed with fresh medium every 2-3 days.
RT-PCR.
RT-PCR was conducted to determine the expression of PAR-1 and the
chloride channels, cystic fibrosis transmembrane conductance regulator
(CFTR), calcium-dependent chloride channel-1 (CLCA-1), and chloride
channel-2 (ClC-2) in SCBN cells. The procedure for RT-PCR was conducted
using the "primer-dropping" method (37) as we
(20) have previously described. Briefly, monolayers of SCBN cells were grown in T25 flasks to near confluence as determined by
light microscopy. Cells were scraped from the flasks and RNA extracted
using 1.5 ml TRIzol reagent per flask. Samples were frozen on dry ice
and stored in TRIzol at
80°C until processed for RNA extraction.
Samples were thawed, and 0.3 ml of chloroform was added to the tube.
Samples were centrifuged at 11,700 g for 30 min at 4°C,
and 500 µl of the aqueous phase were removed and mixed with 750 µl
of isopropanol to precipitate RNA. The samples were centrifuged at
12,000 g for 20 min. The RNA pellet was then washed in 75%
ethanol and centrifuged for 5 min at 7,500 g and redissolved
in 50 µl of ultrapure autoclaved water. The purity and concentration
of the RNA were measured using a Gene Quant II nucleic acid analyzer
(Pharmacia Biotech, Uppsala, Sweden). RNA (2 µg) was added to a
reaction mixture containing 2 µl of 10× PCR buffer, 2 µl of 10 mM
dNTPs, 2 µl N6, and 0.5 µl of RNAguard. Superscript
enzyme (300 U, GIBCO BRL, Burlington, ON, Canada) was added for reverse
transcription. RNA samples were first incubated for 10 min, and the
reaction mixture was heated to 42°C for 50 min and then to 95°C (to
destroy the Superscript enzyme) in a DNA Engine thermal cycler (MJ
Research, Waltham, MA). PCR was performed on either the cDNA from the
RT reaction or RT negative samples to control for contamination
with genomic DNA, using the primer sequences shown in Table
1. PCR for PAR-1 was stopped after 44 cycles (denaturation at 94°C for 17 s, annealing at 53°C for 1 min, and elongation at 72°C for 1 min), for CFTR after 44 cycles
(denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and elongation at 72°C for 1 min), for CLCA-1 after 44 cycles (denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and elongation at 72°C for 1 min), and for ClC-2 after 46 cycles
(denaturation at 94°C for 17 s, annealing at 62°C for 30 s, and elongation at 72°C for 1 min). PCR for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was stopped after 22 cycles (denaturation at 94°C for 30 s, annealing at 55°C for
30 s, and elongation at 72°C for 1 min). PCR products were
purified using the QIAquick PCR purification kit (Qiagen, Mississauga,
ON, Canada) and sequenced by the University of Calgary Core DNA
Service. Sequences were compared with those published on the National
Institutes of Health Genbank database.
Assessment of chloride secretion.
SCBN monolayers were grown to confluence on Snapwell semipermeable
supports. Confluence was determined by the increase in resistance
across the monolayers as measured by an electrovoltohmmeter (EVOM,
World Precision Instruments, Sarasota, FL). Only monolayers with a
resistance >1,000
/cm2 were used. Snapwell supports
were mounted in modified Ussing chambers and bathed on the apical side
with Krebs buffer containing 10 mM mannitol and on the basolateral side
with Krebs buffer containing 10 mM glucose. Krebs buffer contained in
mM: 115 NaCl, 2 KH2PO4, 2.4 MgCl2,
25 NaHCO3, 8 KCl, and 1.3 CaCl2. The
transepithelial potential difference was clamped to 0 V by applying a
short-circuit current (Isc) with a voltage clamp
apparatus (EVC-4000, World Precision Instruments). The change in
Isc was an indicator of the change in the net
electrogenic electrolyte flux across the monolayer.
Isc was recorded with a digital data acquisition
system (MP100, BioPac, San Diego, CA) and analyzed with AcqKnowledge software (version 3.1.3, BioPac).
PAR-1 activation was accomplished with addition of either thrombin or
the PAR-1-activating peptides TFLLR-NH2 (16)
or Ala-parafluoroPhe-Arg-cyclohexyl-Ala-Cit-Tyr-NH2 (Cit-NH2) (19). When used, inhibitors were
added to both the basolateral and apical sides of the monolayers, with
the exception of DIDS, which was added to the apical surface only.
Determination of cAMP.
CFTR intracellular trafficking (31) and channel function
(10) are stimulated by increases in the intracellular
concentration of cAMP. To determine the effects of PAR-1 activation on
cAMP levels, experiments were conducted in which five
75-cm2 flasks of confluent SCBN cells were rinsed with
sterile PBS. Fetal bovine serum-free DMEM (4 ml) was then added to each
flask, and cells were scraped using a cell scraper. Cells and medium were pooled into a 50-ml centrifuge tube, and 1-ml aliquots of cell
suspension were transferred to microfuge tubes. The phosphodiesterase inhibitor B-8279 (Sigma Chemical, Mississauga, ON, Canada) was added to
each tube for a final concentration of 100 µM. Immediately after,
forskolin (10 µM), Cit-NH2 (5 µM), thrombin (5 U/ml),
or the vehicle control HEPES were added and the suspensions allowed to
incubate for 5 min at room temperature. The tubes were then immersed in
liquid nitrogen, cycled three times between liquid nitrogen and a
40°C water bath to lyse the cells, and centrifuged at 15,300 g for 5 min at 4°C. Concentrations of cAMP were determined in supernatants using a commercial ELISA kit (R & D Systems,
Minneapolis, MN) according to the manufacturer's instructions.
Materials.
Thrombin (human; sp act, 2,800 NIH U/mg protein) was purchased from
Calbiochem (San Diego, California). The PAR-1-activating peptides were
synthesized as carboxyamides in-house by the University of Calgary
Peptide Synthesis Facility (directed by Dr. Dennis McMaster).
Thapsigargin was obtained from Alomone (Jerusalem, Israel). Routine
buffer reagents were purchased from BDH (Toronto, ON, Canada). Unless
otherwise specified, all other drugs and reagents were purchased from
Sigma Chemical.
Statistics.
Data are expressed as means ± SE. Comparison of more than two
groups was made by ANOVA with the post hoc Tukey's test using Instat
version 3.00 software (GraphPad Software, San Diego, CA). Comparison of
two groups was made using Student's t-test for unpaired data. P < 0.05 was considered significant.
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RESULTS |
PAR-1 expression and response to activation.
SCBN cells constitutively express PAR-1 mRNA as shown in Fig.
1. The PCR product was sequenced and
compared with the Genbank database, which confirmed human PAR-1
(Genbank accession no. M62424). The level of expression of PAR-1 was
similar to that of the housekeeping gene GAPDH.

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Fig. 1.
Reverse image of RT-PCR gel for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protease-activated
receptor-1 (PAR-1). Lane 1, base pair marker lane;
lane 2, PCR conducted with primers and cDNA obtained from RT
(PAR-1 and GAPDH were present); lane 3, PCR conducted
without cDNA; lane 4, PCR conducted without primers.
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SCBN cells grown to confluence on Snapwell supports exhibited a
baseline Isc of
1 ± 0.3 µA/cm2. Basolateral exposure of monolayers to thrombin or
to the PAR-1-activating peptides Cit-NH2 or
TFLLR-NH2 caused a rapid increase in
Isc that reached a peak within 2 min and
subsequently returned to baseline over the next 5 min (Fig.
2). The maximal change in
Isc observed after basolateral application of
PAR-1 activators was concentration dependent (Fig.
3). Apical application of thrombin caused
no change in Isc, whereas apical application of
the PAR-1-activating peptides caused only slight increases in
Isc, which were much lower than those observed
after basolateral application. The control peptide FSLLR-NH2 (100 µM) did not cause a change in
Isc when added to the basolateral side of SCBN
monolayers (Fig. 2). The monolayers exposed to FSLLR-NH2
responded to subsequent addition of the cAMP-dependent secretagogue
forskolin (10 µM), with an increase in Isc,
indicating that these preparations were still viable. Incubation of
monolayers in chloride-free Krebs buffer significantly reduced the
increases in Isc that occurred after basolateral
application of thrombin or Cit-NH2 (Figs. 2 and
4), suggesting that the change in
Isc observed after PAR-1 activation was due
primarily to stimulation of chloride secretion. A residual increase in
Isc was observed in the presence of
chloride-free buffer, however, and was likely due to bicarbonate ion
transport (6). In addition, the secretory response to
thrombin or Cit-NH2 was reduced by pretreatment of the
monolayers with the nonselective potassium channel blocker barium
chloride (2 mM; Fig. 4).

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Fig. 2.
Representative traces of short-circuit current
(Isc) responses to thrombin and PAR-1-activating
peptides. The y-axis showing absolute
Isc for each trace is included as baselines
varied slightly from one preparation to another. A: exposure
to thrombin, Cit-NH2, and TFLLR-NH2 caused a
rapid, transient increase in Isc. B:
the inactive control peptide FSLLR-NH2 (FS, 100 µM) did
not change Isc. This preparation was still
viable because it responded positively to basolateral application of
forskolin (FSK). C: the increases in
Isc in response to basolateral application of
thrombin or Cit-NH2 were reduced or abolished in
Cl -free Krebs buffer. D: the
Isc responses to thrombin and
Cit-NH2 were mediated through the same receptor, because
treatment with one agonist caused desensitization to subsequent
application of the other agonist. Cell monolayers were still viable as
they responded normally to subsequent basolateral application of
forskolin.
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Fig. 3.
Concentration-response relationships for increases in
Isc induced by activation of PAR-1. Thrombin
(A), Cit-NH2 (B), and
TFLLR-NH2 (C) all caused concentration-dependent
increases in Isc when applied basolaterally. The
response to TFLLR-NH2 was minimal at the highest
concentration tested (100 µM). Responses to apically applied PAR-1
agonists were substantially smaller than those observed after
basolateral activation.
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Fig. 4.
Isc responses of monolayers of
SCBN cells mounted in Ussing chambers to 5 U/ml thrombin and 10 µM
Cit-NH2. A: cells were incubated in normal Krebs
buffer or Cl -free modified Krebs buffer. B:
monolayers were incubated in normal Krebs buffer or Krebs buffer
containing 2 mM BaCl2. * P < 0.05;
** P < 0.01.
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To ensure that the Isc responses observed were
due to activation of PAR-1, studies were conducted in which thrombin or
a PAR-1-AP was added basolaterally to the monolayers to elicit an
increase in Isc. Once baseline
Isc had been reestablished (5-8 min),
thrombin or the PAR-1-AP was added basolaterally again to assess
desensitization. Activation of PAR-1 with either thrombin or an
activating peptide causes rapid desensitization of the receptor and,
hence, unresponsiveness to subsequent attempts to elicit a
PAR-1-dependent response (32). Exposure to a PAR-1
activator caused almost complete desensitization to a subsequent
application of the same PAR-1 activator (Fig. 5). Furthermore, activation of PAR-1
caused crossdesensitization to subsequent application of a different
PAR-1 activator (Figs. 2 and 5).

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Fig. 5.
Isc responses of monolayers of SCBN cells
mounted in Ussing chambers to Cit-NH2 (Cit; 5 µM),
TFLLR-NH2 (TF; 25 µM), or thrombin (Thr; 5 U/ml). Cells
were not pretreated (control; C) or were pretreated with the same PAR-1
activator to demonstrate autodesensitization or with a different PAR-1
activator to demonstrate crossdesensitization. * P < 0.05; ** P < 0.01; *** P < 0.001.
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Calcium dependency of PAR-1-induced chloride secretion.
PAR-1 activation leads to a rapid and transient increase in
intracellular calcium (16). To determine if the chloride
secretion induced by PAR-1 activation was due to increased
intracellular calcium concentration, we first determined if PAR-1
activation caused increased cytosolic calcium in SCBN monolayers grown
on coverslips and loaded with the calcium indicator fluorophore fura 2. Figure 6 shows representative micrographs
with a fluorescence ratio of 1 × 105 cells/monolayer
exposed to thrombin (50 U/ml) or Cit-NH2 (100 µM). Both
thrombin and Cit-NH2 caused an increase in fluorescence ratio. However, not all cells responded with an increase in
fluorescence. Both thrombin and Cit-NH2 caused
crossdesensitization to the other PAR-1 agonist (Fig. 6). The higher
concentration of agonist required to activate PAR-1 in this system,
compared with the chloride secretion experiments, may be due to the
monolayers being grown on glass coverslips. This would limit access of
apically added compounds to the basolateral surface of the cells, where
the receptor is likely located.

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Fig. 6.
Ca2+ signaling responses in SCBN cells grown
on coverslips and loaded with fura 2. A: representative
traces of the change in fluorescence ratio after PAR-1 activation. Both
thrombin and Cit-NH2 increased the intracellular
concentration of Ca2+ and caused crossdesensitization to
the other PAR-1 agonist. B: representative micrographs of
SCBN cells grown on glass coverslips and loaded with fura 2. Increasing
brightness represents computer-generated images of increasing
fluorescence ratio. Brighter areas indicate increased Ca2+
in response to thrombin (50 U/ml) or Cit-NH2 (100 µM).
The 0-s time point indicates the fluorescence ratio immediately before
addition of the PAR-1 activator.
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To determine if the increases in calcium elicited by PAR-1 activation
were coupled to chloride secretion, we conducted experiments with SCBN
monolayers grown on Snapwell supports and mounted in Ussing chambers.
The responses to thrombin and Cit-NH2 were assessed in the
presence of the intracellular calcium reuptake inhibitor thapsigargin
(250 nM), the L-type calcium channel blocker verapamil (10 µM), or
DIDS (300 µM), which blocks CLCA, but not CFTR. Only thapsigargin
reduced the responsiveness of the SCBN cells to thrombin or
Cit-NH2 compared with vehicle controls (Fig.
7). Neither DIDS nor verapamil affected
the CFTR-dependent response to forskolin. Thapsigargin caused a
prolonged increase in Isc, and subsequent exposure to thrombin resulted in a decrease in
Isc. Thapsigargin pretreatment did not affect
the response to forskolin (Fig. 7).

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Fig. 7.
Effect of Ca2+ blockers on the
Isc response to thrombin (5 U/ml; A)
or Cit-NH2 (10 µM; B) by SCNB cell monolayers
in Ussing chambers. Monolayers were treated with drug 20-30 min
before addition of either thrombin or Cit-NH2.
C: representative trace of Isc
responses in the presence of thapsigargin. Thapsigargin caused an
increase in Isc and reversed the response to
subsequent exposure to thrombin but did not affect the response to
forskolin. Con, control; Thaps, thapsigargin; Verap, verapamil.
** P < 0.01; *** P < 0.001.
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Chloride transport in intestinal epithelial cells can occur through
several channels. Apically directed chloride transport occurs through
either the CFTR, which is modulated through cAMP, or CLCA. Furthermore,
ClC-2 has recently been implicated in vectorial chloride secretion by
intestinal epithelial cells (22). Because the SCBN cell
monolayers responded to the cAMP-dependent secretagogue forskolin and
because the Isc response to PAR-1 activation was calcium dependent, we assumed that both chloride conductance pathways were present in SCBN cells. However, because the channel responsible for chloride secretion in SCBN cells has not been characterized, we
conducted RT-PCR to look for expression of CFTR, ClC-2, and CLCA-1. PCR
products were purified using the Qiagen PCR purification kit and
sequenced at the University of Calgary Core DNA Service. Sequences of
amplified DNA were compared with those available in the Genbank
database. The results of RT-PCR indicate that SCBN cells express mRNA
for both CFTR and ClC-2. Furthermore, when PCR was performed on
RT-negative samples to control for possible contamination with genomic
DNA, no product was found. We were unable to show CLCA-1 mRNA
expression via this method (Fig. 8).

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Fig. 8.
Reverse image of RT-PCR for cystic fibrosis transmembrane
conductance regulator (CFTR), Ca2+-dependent
Cl channel-1 (CLCA-1), Cl channel-2
(ClC-2), and GAPDH in SCBN cells. A: CFTR was expressed
in these cells, whereas CLCA-1, expected at 509 bp based on our primer
sequence, was not; , the negative control for CFTR, ruling out
contamination with genomic DNA. B: SCBN cells express
ClC-2 mRNA; , negative control.
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To rule out a role for cAMP-dependent transport in the
Isc response to PAR-1 activation, we conducted
further experiments in which SCBN cells were exposed to vehicle,
thrombin, Cit-NH2 or forskolin and subsequently assayed for
cAMP using ELISA. PAR-1 activation caused no increase in cAMP compared
with vehicle control. In contrast, exposure of the cells to forskolin
caused a large increase in cAMP levels in these cells (Fig.
9).

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Fig. 9.
Adenylate cyclase activity assessed in SCBN cells after
exposure to HEPES buffer (control), thrombin (Throm, 5 U/ml),
Cit-NH2 (10 µM), or forskolin (10 µM). Activity is
represented as the generation of cAMP in the presence of a
phosphodiesterase inhibitor. PAR-1 activation failed to activate
adenylate cyclase. * P < 0.05.
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DISCUSSION |
In the present study, we have shown that PAR-1 is expressed in a
nontransformed, human small intestinal epithelial crypt cell line
(SCBN) and that activation of PAR-1 on the basolateral surface of SCBN
cells results in apically directed chloride secretion. Through the use
of RT-PCR and selective PAR-1-activating peptides, we have shown that
it is indeed PAR-1, and not a different PAR, being activated by
thrombin. Furthermore, we have shown that PAR-1 activation leads to
chloride secretion through a mechanism that is dependent on release of
calcium from intracellular stores and independent of adenylate cyclase activity.
Four PARs have been cloned to date. Of these, PAR-1 and PAR-4 in
conjunction with PAR-3 are activated by thrombin whereas PAR-2 is
activated by trypsin and perhaps tryptase. An important role for PARs
is suggested by their wide tissue distribution. For example, PAR-1 is
expressed in human platelets, endothelial cells, fibroblasts,
monocytes, T cell lines, osteoblast-like cells, smooth muscle cells,
neurons, and glial cells in the brain and periphery and certain tumor
cell lines (7). Our study is the first to demonstrate
PAR-1 expression in the intestinal epithelium and to link its
activation to ion transport.
We chose the SCBN duodenal epithelial cell line because it is
nontransformed, derived from humans, and capable of apically directed
chloride transport and it forms electrically tight monolayers, thus
making it amenable to study in the Ussing chamber (25). In
our hands, SCBN monolayers consistently demonstrated electrical resistances of >2,000
/cm2 (data not shown). The SCBN
cell line also expresses PAR-1 mRNA as shown by RT-PCR. Sequencing of
the PCR product showed identity with the sequence in the Genbank
database (accession no. M62424), confirming that our product was indeed
human PAR-1. Furthermore, in Ussing chamber studies in which the SCBN
monolayers were studied under voltage clamp conditions, application of
thrombin or the PAR-1 activating peptides Cit-NH2 and
TFLLR-NH2 caused a rapid increase in
Isc when applied to the basolateral side of the
monolayers and almost no response when applied apically, suggesting
that PAR-1 expression is polarized and restricted to the basolateral aspect of the cells. The fact that there was a slight
Isc response to apical application of the
PAR-1-activating peptides suggests that either the peptides were able
to translocate across the epithelium to activate basolateral PAR-1 or
there is a sparse population of apical PAR-1. PAR-1 localization in
renal proximal tubular epithelium is also polarized and basolateral
(13). The Isc changes observed
after PAR-1 activation were due to the movement of chloride ion,
because responses were significantly reduced in chloride-free buffer.
The residual increase in Isc seen in the absence
of chloride was likely due to bicarbonate secretion (6).
Reduction of the secretory response to PAR-1 activation by inhibition
of potassium conductance using barium chloride also confirmed the
involvement of the basolateral potassium channel, which is involved in
maintaining the energetics favorable to chloride transport
(2).
Having demonstrated that PAR-1 activation could stimulate vectorial
chloride transport, we next sought to determine which chloride channel
was involved. Intestinal epithelial cells express a number of chloride
channels. The channels involved in apical, outwardly directed chloride
currents are CFTR and CLCA (2, 9). Furthermore, ClC-2,
which in intestinal cells has previously been linked to volume
regulation, has recently been shown (22) to have a
polarized distribution in the cells and to contribute to chloride
currents. CFTR activation is controlled by several intracellular
signaling pathways that control channel phosphorylation and
dephosphorylation (11). Conspicuous among these is a
cAMP-dependent pathway that stimulates CFTR trafficking to the apical
membrane (31) and channel opening (11). SCBN
cells respond to forskolin (25), which activates adenylate
cyclase, suggesting that CFTR is present in these cells. In this study,
we have confirmed the responsiveness of this cell line to forskolin and
shown that CFTR mRNA is expressed. The latter was determined by RT-PCR
and confirmed by sequencing of the PCR product (Genbank accession no.
NM000492). However, despite the presence of CFTR in this cell line, the
response to PAR-1 activation is not due to a CFTR-mediated chloride
current. PAR-1 activation failed to increase intracellular cAMP
concentrations, whereas forskolin caused a 70-fold increase.
Furthermore, inhibition of calcium-dependent signaling pathways blocked
the response to PAR-1 activation but did not affect the response to forskolin.
Activation of calcium-dependent pathways is a hallmark of
PAR-1-initiated signaling (16, 19). Using fura 2-labeled
SCBN cells, we showed that PAR-1 activation caused a rapid, transient increase in intracellular calcium that followed a time course similar
to that observed for the increase in Isc in ion
transport studies. The transient nature of the calcium signaling
response is characteristic of PAR-1 activation (16, 19).
Not all cells responded to PAR-1 activators. This could be due to a
heterogeneous population of SCBN cells, in which only a subset of cells
express PAR-1, or to lack of accessibility of the activator to the
basolaterally located receptor. The fact that larger concentrations of
thrombin and Cit-NH2 were required to elicit a calcium
signal compared with those required to stimulate changes in
Isc can be explained by the latter point.
Furthermore, we showed that PAR-1-mediated chloride secretion in SCBN
cells was calcium dependent because it was inhibited by thapsigargin.
The fact that the chloride secretory response to PAR-1 activation is
reduced by depletion of intracellular calcium stores with thapsigargin,
and not by blocking the entry of extracellular calcium with verapamil,
suggests that intracellular reserves are being used in the signaling
process. As expected, thapsigargin on its own stimulated an increase in
Isc, likely due to the initial liberation of
calcium from intracellular stores. Interestingly, pretreatment with
thapsigargin resulted in a reversal of the current response to
subsequent PAR-1 activation. We speculate that removal of stored
calcium uncovers an additional signal transduction pathway that
inhibits chloride secretion, but which is masked by the larger
stimulatory signal present when intracellular calcium is available.
However, a thorough investigation of this inhibitory pathway was beyond
the scope of this study.
The calcium dependency of PAR-1-induced chloride secretion suggested
that one of the members of the CLCA family was involved. CLCA-1 is the
isoform that is expressed in human small intestine (14).
However, we were unable to detect the presence of human CLCA-1 by
RT-PCR using previously published (14) primers and conditions. Nevertheless, this does not dismiss the possibility that
other chloride channels of this type, distinct from human CLCA-1, are
present in SCBN cells. New members of the calcium-activated chloride
channel family have recently been characterized (1, 9) and
more will undoubtedly be cloned. CLCA can be demonstrated pharmacologically by inhibition with DIDS (14). In our
study, PAR-1-mediated chloride secretion is not inhibited by apical
DIDS. Taken together, the weight of evidence points to the selective activation of a chloride channel distinct from CFTR and CLCA by PAR-1 activation.
It is possible that ClC-2 is involved in PAR-1-induced chloride
secretion in this cell line. Mohammad-Panah et al. (22) have shown that ClC-2 is expressed in human intestinal epithelial cells
and that this distribution may be polarized. Using RT-PCR, we showed
that SCBN cells express ClC-2. Further study is necessary to show
unequivocally that ClC-2 mediates the chloride secretory response to
PAR-1 activation.
The presence of PAR-1 on intestinal epithelial cells has important
implications for barrier function during tissue injury. The intestinal
epithelium provides an important barrier to the translocation of
luminal pathogens or antigens to the mucosal lamina propria
(4). This barrier consists of tight junctions preventing
paracellular permeation of luminal constituents, plus the ability of
crypt cells to secrete chloride and water (4). A
compromised barrier allows access of luminal contents to the lamina
propria, which in turn may initiate, exacerbate, or perpetuate inflammation. Thrombin has been shown to play a role in the
inflammatory response, including that which characterizes inflammatory
bowel disease (27). The epithelium, particularly the
basolateral surface, would be exposed to thrombin during
inflammation-associated hemorrhage. Two scenarios can be envisioned for
the interaction of thrombin with epithelial PAR-1 in vivo. First, PAR-1
activation could lead to a secretory response that could contribute to
the diarrhea symptomatic of inflammatory conditions of the gut.
Conversely, PAR-1 activation during inflammation may represent an
activation of the secretory component of the epithelial host defense function.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by grants from the Canadian
Institutes of Health Research (formerly the Medical Research Council of
Canada; M. D. Hollenberg, J. L. Wallace, and W. K. MacNaughton) and the Natural Sciences and Engineering Research Council
(A. Buret). M. C. Buresi is funded by an Alberta Heritage
Foundation for Medical Research studentship and N. Vergnolle by the
University of Calgary NicOx Chair in Inflammation Research. W. K. MacNaughton is an Alberta Heritage Foundation for Medical Research
Scholar. J. L. Wallace is an Alberta Heritage Foundation for
Medical Research Senior Scientist and a Canadian Institutes of Health
Research Senior Scientist.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: W. MacNaughton, Mucosal Inflammation Research Group, Univ. of Calgary, 3330 Hospital Dr NW, Calgary, Alberta, T2N 4N1 Canada (E-mail: wmacnaug{at}ucalgary.ca).
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 15 December 2000; accepted in final form 19 March 2001.
 |
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