1 Universitäts-Kinderklinik, Albert-Ludwigs-Universität Freiburg, 79106 Freiburg, Germany; and 2 Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland 4072, Brisbane, Australia
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ABSTRACT |
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Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial
cells and rat jejunum. With the use of a microUssing chamber, we
demonstrate the role of PAR-2 for ion transport in native human colonic
mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl secretion when added to the
basolateral but not luminal side of normal epithelia. Activation of
Cl
secretion by trypsin was inhibited by indomethacin and
was further increased by cAMP in normal tissues but was not present in
CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl
, by basolateral bumetanide, and in the presence
of barium or clotrimazole, but not by tetrodotoxin. Furthermore,
trypsin-induced secretion was inhibited by the Ca2+-ATPase
inhibitor cyclopiazonic acid and in low-Ca2+ buffer. The
effects of trypsin were almost abolished by trypsin inhibitor.
Thrombin, an activator of PAR types 1, 3, and 4, had no effects on
equivalent short-circuit currents. The presence of PAR-2 in human colon
epithelium was confirmed by RT-PCR and additional experiments with
PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte
secretion by release of mast cell tryptase and potentiation of PAR-2
expression by tumor necrosis factor-
may contribute to the
hypersecretion observed in inflammatory processes such as chronic
inflammatory bowel disease.
protease-activated receptors; ion transport; trypsin; cystic fibrosis transmembrane conductance regulator; inflammatory bowel disease
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INTRODUCTION |
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SERINE PROTEASES HAVE RECENTLY been demonstrated to act as signaling molecules (12). They regulate cells by specifically cleaving and activating members of a new family of protein-activated receptors (PARs). Apart from the three types of thrombin receptors, PAR-1, -3, and -4, the subtype PAR-2 has been identified as a receptor for trypsin and mast cell tryptase (12, 27). PARs are G protein-coupled receptors that are activated either by soluble ligands that reversibly bind to the receptor or by irreversible cleavage by a protease. In the latter case, a serine protease will cleave the receptor at the extracellular NH2-terminal end and a tethered ligand, which is now exposed, becomes available and binds to the receptor (12). PAR-2 was cloned initially from a mouse genomic library and subsequently from human kidney cDNA (5, 27). The receptor shows a wide organ distribution and is highly expressed in pancreas, kidney, colon, liver, small intestine, and airways (5, 7, 10). Because little is known about the function of PAR-2 in the human colon, we examined in the present study the effects of trypsin and PAR-2-activating peptide (AP) in human rectal biopsies.
The function and physiological significance of PAR-2 is still poorly
understood. However, recent studies suggested that PAR-2 might
participate in the control of ion transport in gastrointestinal epithelia. Thus PAR-2 has been implicated in activating ion transport in cultured dog pancreatic duct cells (26). These studies
suggested an activation of luminal Cl and basolateral
K+ channels in pancreatic epithelial cells due to
stimulation of basolaterally located PAR-2. Such an activation of ion
secretion may promote clearance of toxins and debris from the
pancreatic duct. Thus pancreatic trypsin may activate PAR-2, which are
located in the pancreatic duct under both physiological and
pathophysiological conditions. PAR-2 have also been identified in both
luminal and basolateral membranes of epithelial cells in the small
intestine (21). Expression of these receptors has been
demonstrated by Northern blot analysis and immunohistochemistry
(10, 21), and previous data obtained from rat jejunal
mucosa suggested activation of ion transport by trypsin
(33). On the basis of considerable differences in agonist
concentrations and potency profiles for activation of short-circuit
currents, the authors concluded that a receptor different than PAR-2 is
activated by trypsin in the rat intestine (33).
However, the nature of ion conductances that are activated by stimulation of intestinal PARs and the responsible intracellular second-messenger pathways are still largely unknown. In the present study, we explored these questions by using a modified Ussing-chamber technique, which allows measurement of ion transport in small human rectal biopsies. The results presented here indicate pronounced activation of ion transport in the human colon by trypsin. Electrolyte secretion could be elicited repeatedly by trypsin in the same mucosal biopsy. It is thus very likely that activation of PARs, present in basolateral membranes of human colonic epithelial cells, contributes to the hypersecretion found in inflammatory processes such as chronic inflammatory bowel disease (13).
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MATERIALS AND METHODS |
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Patients.
Ussing-chamber measurements were performed on rectal mucosa biopsies
obtained from 30 normal individuals and 11 cystic fibrosis (CF)
patients. All CF subjects presented with pancreatic insufficiency and
chronic lung disease and fulfilled the diagnostic criteria of CF,
including elevated sweat tests (30). Genotyping by DNA analysis of all CF patients showed that five were homozygous and six
were heterozygous for F508-CF transmembrane conductance regulator (CFTR; allelic frequency of 72%). Testing of an additional panel of
the 19 most prevalent CFTR mutations among the Caucasian population in
Europe, including G542X, N1303K, 1717-1 G>T, W1282X, G551D, R553X, R1162X, R334W, R117H, 621+1G>T, 3849+10kbC>T, 3659delC, 1078delT, R347P, A445E, S1251N,
I507, 2183AA>G, and E60X (ELUCIGENE CF20; AstraZeneca Diagnostics) failed to identify the second disease causing mutation in six CF patients. Small superficial tissue biopsies
were obtained by rectoscopy and forceps biopsy performed at the
University Children's Hospital Freiburg. The study was approved by the
ethical committee, and the patients had given written informed consent.
For children under the age of 18 yr, parents obtained detailed
information and gave signed informed consent.
Ussing-chamber experiments.
Rectal tissue biopsies were immediately stored in an ice-cold buffer
solution of the following composition (in mM): 127 NaCl, 5 KCl, 5 D-glucose, 1 MgCl2, 5 Na pyruvate, 10 HEPES,
and 1.25 CaCl2 and 10 g/l albumin. Tissues were mounted
into a perfused microUssing chamber with a circular aperture of 0.95 mm2 as described previously (24). In brief,
the luminal and basolateral sides of the epithelium were perfused
continuously at a rate of 10 ml/min (chamber volume, 1 ml). The bath
solution had the following composition (in mM): 145 NaCl, 0.4 KH2PO4, 1.6 K2HPO4, 5 D-glucose, 1 MgCl2, and 1.3 Ca-gluconate, and
pH was adjusted to 7.4. For low Cl solutions,
Cl
was reduced to 5 mM by equimolar replacement of 142 mM
NaCl by Na+ gluconate, and 4 mM Ca gluconate was added to
compensate for the Ca2+-chelating effects of gluconate.
Bath solutions were heated by a water jacket to 37°C. Experiments
were carried out under open-circuit conditions. Transepithelial
resistance (Rte) was determined by applying
short (1 s) current pulses (
I = 0.5 µA), and the
corresponding changes in transepithelial voltage (Vte;
Vte) and basal Vte were recorded
continuously. Values for the Vte were referred to the serosal side of the epithelium. Voltage deflections obtained under conditions without the mucosa present (
V'te) were
subtracted from those obtained in the presence of the tissues.
Rte was calculated according to Ohm's law
[Rte = (
Vte
V'te)/
I]. The equivalent short-circuit current (Ieq) was determined from
Vte and Rte, i.e., Ieq = Vte/Rte. Tissues were allowed
to equilibrate for 30 min before basal bioelectric properties were taken.
Cell culture, RNA isolation, and RT-PCR.
HT29 and T84 colonic carcinoma cells were grown
in culture as described previously (18). In brief, cells
were grown in Dulbecco's modified Eagle's medium with (in mM) 10 Na+-Hepes buffer, 4 L-glutamine (with 0.04 g/l
penicillin), 0.09 streptomycin, and 100 newborn calf serum in 5%
CO2. Total RNA was isolated from superficial biopsies of
rectal mucosa and from the human colonic cell lines HT29
and T84 using RNeasy spin columns (Quiagen, Hilden, Germany), as
described previously (22), and was reverse transcribed at
37°C for 1 h using random primer and RT (Superscript RT, Life
Technologies). The size of the expected 543-bp fragment of PAR-2 was
amplified by PCR using the sense primer 5'-GTGTTTGTGGTGGGTTTGCC-3' and
antisense primer 5'-CATCAGCACATAGGCAGAGG-3' (94°C for 60 s, 35 cycles of 94°C for 1 min, 57°C for 30 s, 72°C for 60 s). PCR products were visualized by loading an 8-µl sample on a 0.9%
agarose gel using a 123-bp marker as a standard. The PCR product was
subcloned into pBluescript SK () vector and sequenced using Thermo
Sequenase I (Pharmacia) and a 373A DNA sequencer (Applied Biosystem).
Compounds and statistics. Amiloride, bumetanide, indomethacin, cyclopiazonic acid (CPA) IBMX, forskolin, TTX, trypsin (bovine, 9,820 U/mg protein), trypsin inhibitor (type III-O chicken egg white; 1 mg will inhibit 1.1 mg trypsin with an activity of 10,000 BAEE U/mg protein), and thrombin (bovine, 56 U/mg protein) were all obtained from Sigma (Deisenhofen, Germany). PAR-2-AP (SLIGRL-NH2) corresponding to the tethered ligand of mouse PAR-2 and the reverse peptide (RP; LRGILS-NH2) were synthesized by solid-phase methods and purification by high-pressure liquid chromatography (Big Biotech, Freiburg, Germany). All used chemicals were of highest grade of purity available. From some individuals, transepithelial measurements were performed on more than one tissue sample. When multiple samples were studied by the same protocol, data were averaged to obtain a single mean value for each individual subject. Continuous bilateral bath perfusion allowed to perform consecutive measurements under different experimental conditions on the same tissue, and all experiments were performed in a paired fashion, in which each tissue served as its own internal control. Data for transepithelial measurements are shown as original recordings or as means ± SE and are generally reported as peak responses of Vte and Isc (n = number of subjects). Statistical analysis was performed using paired Student's t-test. Data obtained from CF and non-CF tissues were compared by unpaired Student's t-test. P values <0.05 were accepted to indicate statistical significance.
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RESULTS |
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PAR-2 are expressed in native human colonic epithelium and are
activated by trypsin.
RNA was prepared from superficial biopsies of rectal mucosa and from
the human colonic cell lines HT29 and T84, and
RT-PCR was performed using primers specific to the sequence of human PAR-2. A 543-bp fragment was obtained, which was verified as a PAR-2
fragment by subsequent cloning and sequencing (Fig.
1). This result confirmed expression of
PAR-2 for distal colon and rectum, which has been detected recently by
immunocytochemistry (10) and Northern blot analysis in rat
colon (21).
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Cl secretion is not activated by thrombin and is
inhibited by trypsin inhibitor.
PAR activation requires specific enzymatic cleavage by trypsin or
tryptase, which releases a tethered ligand that activates its own
receptor (12). It has been shown that trypsin-mediated activation of PAR-2 on epithelial cells can be abolished by
preincubation of trypsin with trypsin inhibitor
(26). To investigate whether the effects of
trypsin on ion transport in human rectal biopsies could be antagonized
by trypsin inhibitor, we compared the effects of trypsin in the absence
and presence of trypsin inhibitor (1:1). After trypsin was pretreated
with the inhibitor, the effects of trypsin on
Ieq were attenuated significantly from
56.8 ± 11.6 to
11.8 ± 5.6 µA/cm2
(
Vte was reduced from
1.0 ± 0.1 to
0.2 ± 0.1 mV; n = 4; Fig. 4,
A and B). Trypsin has been shown to act on
several PARs in the rank order PAR-2
PAR-4 > PAR-1 = PAR-3, whereas thrombin is a strong activator of PAR-1, PAR-3, and
PAR-4 (12). To further characterize the PAR involved in
trypsin-induced ion transport in the human distal colon, the effects of
both trypsin and thrombin were compared. As depicted in Fig. 4,
C and D, thrombin (1 µM, basolateral) failed to
induce Cl
secretion, whereas trypsin activated
lumen-negative responses of Vte and
Ieq in paired experiments. Together, the present
experiments strongly suggest that trypsin acts on basolateral PAR-2 in
the human distal colon.
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Trypsin-induced Cl secretion depends on
cAMP-activation and requires luminal CFTR Cl
channels.
We previously demonstrated that Ca2+-mediated
Cl
secretion induced by cholinergic stimulation of human
distal colon relies on functional CFTR as the luminal Cl
channel (24). However, previous results obtained on
cultured cells from the kidney collecting duct and pancreatic duct
suggest activation of Ca2+-dependent Cl
channels by stimulation of PAR-2 (2, 26). Given these
results, we asked whether trypsin may activate otherwise dormant
non-CFTR Cl
channels in human rectal mucosa. To that end,
we examined the effects of trypsin in the absence and presence of
cAMP-dependent stimulation and compared the responses obtained in
tissues from normal individuals and CF patients. The effects of trypsin
were compared with the effects of carbachol (100 µM, basolateral) in a strictly paired fashion under three different conditions:
1) under basal conditions, 2) after perfusion
with indomethacin, and 3) after cAMP-dependent stimulation
with IBMX (100 µM) and forskolin (1 µM, both basolateral). As shown
in Fig. 5A, trypsin-induced anion secretion required coactivation by cAMP. Under baseline conditions with variable CFTR activity, trypsin induced a
lumen-negative secretory response of
66.6 ± 22.8 µA/cm2 (
Vte =
1.3 ± 0.5 mV;
n = 10). Treatment with the cyclooxygenase inhibitor
indomethacin inhibits prostaglandin synthesis, including the formation
of PGE2, which has been identified as a major endogenous agonist of cAMP-dependent Cl
secretion in human colon.
Indomethacin (10 µM, basolateral, 60 min) pretreatment abolished
trypsin-induced ion transport almost completely
(
Vte =
0.1 ± 0.1 mV;
Ieq =
4.8 ± 2.2 µA/cm2; n = 10), whereas subsequent
activation with IBMX and forskolin induced a sustained secretory
response and resulted in a significant increase in trypsin-activated
anion secretion (
Vte =
1.4 ± 0.4 mV;
Ieq =
99.8 ± 29.5 µA/cm2; n = 10) (Fig. 5, A and
C). Ion-substitution experiments showed that cAMP and
trypsin-induced secretory responses were carried by Cl
.
In the presence of IBMX and forskolin, replacement of extracellular Cl
by gluconate (bilateral equimolar replacement of 142 mM Cl
by gluconate; 5 mM Cl
remaining)
resulted in a significant inhibition of Vte and
Ieq (
Vte = 1.5 ± 0.2 mV;
Ieq = 46.9 ± 15.0 µA/cm2; n = 4). Furthermore,
trypsin-mediated secretion was almost abolished in the presence of low
Cl
buffer (trypsin response under normal
Cl
:
Vte =
2.6 ± 0.3 mV;
Ieq =
80.0 ± 23.8 µA/cm2 vs. trypsin response under low Cl
:
Vte =
0.2 ± 0.1 mV;
Ieq =
2.2 ± 0.3 µA/cm2; n = 4), demonstrating that PAR-2
activation induces Cl
secretion in normal human colon. In
contrast, in tissues obtained from CF patients (n = 10), trypsin failed to induce Cl
secretion under all
experimental conditions. Instead, trypsin activated a lumen-positive
K+ secretory response, which was +12.2 ± 4.5 µA/cm2 (
Vte = 0.4 ± 0.1 mV;
n = 10) in the presence of indomethacin. These
experiments demonstrate that trypsin-induced Cl
secretion
depends on the presence of functional CFTR as the luminal Cl
channel. Although the magnitude of cholinergic
secretion in both normal and CF tissues was significantly larger
compared with trypsin-induced secretion, both agonists demonstrated a
very similar behavior. This observation may suggest that both agonists
share a common intracellular signal-transduction pathway.
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Trypsin-induced Cl secretion requires basolateral
Cl
uptake and activation of
K+ channels.
In the presence of amiloride, lumen-negative responses of
Vte and Ieq are caused by anion
secretion. Due to the lack of highly specific inhibitors of CFTR
Cl
channels, we used bumetanide (100 µM, basolateral),
an inhibitor of the Na+-K+-2 Cl
cotransporter, to block basolateral Cl
uptake. As shown
in Fig. 6, both cAMP-activated and
trypsin-induced anion secretion was almost completely abolished by
bumetanide, indicating that trypsin activated transepithelial
Cl
secretion.
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PAR-mediated ion transport requires an increase in intracellular
Ca2+.
Activation of PAR-2 by trypsin has been shown to increase intracellular
Ca2+ and generation of PGE2 (12).
The present results suggested that ion transport activated by
stimulation of PAR-2 is caused by increase in intracellular
Ca2+. To further confirm the role of Ca2+ as
the mediator of PAR-2 effects in human colon, tissues were treated for
20 min with the Ca2+-ATPase inhibitor CPA (50 µM, both
sides). Inhibition of ATPase-dependent Ca2+ reuptake into
endoplasmic stores is expected to cause a transient increase in
intracellular Ca2+, followed by inhibition of
agonist-induced store release. Accordingly, addition of CPA induced an
initial Cl secretory response of
62.8 ± 13.9 µA/cm2 (
Vte =
0.6 ± 0.1 mV;
n = 5). After 20 min of CPA treatment, a stable plateau
was reached, which was not different from precontrol values. As shown
in Fig. 7, A and B,
the effect of trypsin was almost completely abolished in the presence
of CPA. Similar to the trypsin response, cholinergic stimulation by
carbachol (CCH) was also inhibited in the presence of CPA (Fig.
7, C and B). To address the role of extracellular
Ca2+ in PAR-2-mediated secretion, we compared the effect of
trypsin in low (1 µM) and normal Ca2+ (1.3 mM) bath
solutions. Bilateral perfusion with 1 µM Ca2+ had no
effect on Vte or Ieq. In the
presence of low-Ca2+ buffer, the effect of trypsin was
almost abolished (trypsin response under normal Ca2+:
Vte =
1.2 ± 0.2 mV;
Ieq =
56.5 ± 7.9 µA/cm2 vs. trypsin response under low Ca2+:
Vte =
0.3 ± 0.1 mV;
Ieq =
23.2 ± 11.8 µA/cm2; n = 3). Similar observations were
obtained when tissues were stimulated with CCH in normal compared with
low Ca2+ solution (data not shown). These data suggest that
activation of PAR by trypsin and stimulation of Cl
secretion in the human colon depends on intracellular Ca2+
signaling by endoplasmic stores release and extracellular
Ca2+ entry.
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DISCUSSION |
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Trypsin can act as a signal molecule that specifically regulates
cells by cleaving and activating PAR-2 (12). Trypsin has previously been shown to regulate ion transport in cultured epithelial cells from the pancreatic duct and kidney collecting duct by acting on
PAR-2 located on the basolateral side of the epithelium (2, 26). Whereas a physiological role of PAR-2 detected in cultured kidney cells remains obscure, a protective function during pancreatitis was claimed for PAR-2 expressed on the basolateral side of pancreatic duct cells. A recent report elucidated the role of PAR-2 in regulating salivary and pancreatic exocrine secretion in vivo in rats and in mice
(20). A similar protective role of PAR-2 has been
suggested for the airways (7): trypsin/trypsinogen is
released from the airways and binds to PAR-2 coexpressed in airway
epithelial cells, which then leads to paracrine release of a
cyclooxygenase product, most likely PGE2, which induces
bronchorelaxation (7). This study also analyzed the time
course of recovery for responsiveness to PAR-2 activation after trypsin
desensitization. It was found that a second trypsin response could be
induced as early as 15 min after the initial application of trypsin and
that trypsin-mediated relaxation recovered almost completely after 45 min. Resensitization could be blocked by brefeldin A and cycloheximide,
indicating a fast de novo synthesis and trafficking of preformed
receptors. The present results suggest that PAR-2 have a similarly high
turnover rate in human distal colonic epithelium, because we were able to induce Cl secretion repeatedly only 20-40 min
after the last application of trypsin. Protease-activated receptors
have also been examined in cultured human bronchial epithelial cells.
It was found that stimulation of PAR-2 transiently increased
Cl
secretion, which was followed by a sustained
inhibition of amiloride-sensitive short-circuit currents
(11). It is therefore likely that PAR-2 play a role in
regulation of Na+ absorption and Cl
secretion
in human airways.
A basolateral PAR has been implicated in trypsin-mediated activation of
ion transport in rat jejunum (33). From differences in the
potency profile of the originally described AP (SLIGRL-NH2) and an alternative PAR-2-selective AP (LIGRLO-NH2), the
authors suggested that a receptor different from PAR-2 is activated by trypsin in rat intestine. However, such a receptor has not yet been
identified. It cannot be ruled out that 1) different PAR-2 subtypes exits that demonstrate different potency profiles for these
synthetic agonists or that 2) the binding affinity of PAR-2 for different peptides is modulated by coexpression of other associated membrane proteins in the native epithelium. PAR-2 expression was detected in both basolateral and luminal membranes of enterocytes of
the rat jejunum in another study (21). Stimulation of
cultured enterocytes by basolaterally applied trypsin or
PAR-2-activating peptide induced a release of arachidonic acid,
generation of inositol 1,4,5-trisphosphate, and production and
secretion of PGE2 (21). Thus pancreatic
trypsin in concentrations usually present in the lumen of the jejunum
may be able to activate PAR-2 located in either luminal or basolateral
membranes of enterocytes (14) The data shown in the
present study demonstrate that basolateral PAR-2 are responsible for
trypsin-mediated activation of Cl secretion in human
distal colon. Because trypsin had no effect on ion transport when
applied from the luminal side, the receptors mediating
trypsin-activated ion secretion are strictly located on the basolateral
side of the epithelium. Differences in luminal and basolateral PAR-2
expression in small intestine and distal colon may indicate different
physiological functions but could also be due to species differences.
Importantly, pretreatment of tissues with TTX had no effect on
trypsin-mediated Cl
secretory responses, indicating that
PAR-2 is expressed on the basolateral membrane of epithelial cells
rather than on subepithelial structures, e.g., neuronal fibers. To
assess trypsin-mediated Cl
secretion, experiments were
generally performed in the presence of amiloride to inhibit
electrogenic Na+ absorption. However, we made
quantitatively similar observations when trypsin was added under basal
conditions in the absence of amiloride. Although it cannot be excluded
that the increase in Vte and Ieq
observed in the absence of amiloride is related to activation of cation
absorption, the lumen-negative trypsin response in the absence of
amiloride would also be in agreement with anion secretion under
physiological conditions. This is well conceivable, because colonic
crypts are composed of functionally distinct compartments, with
Cl
secretion predominantly taking place at the crypt
basis and Na+ absorption at the surface epithelium
(15). Experiments using trypsin concentrations
100 nM
indicate that that PAR-2 activation may actually inhibit anion
secretion in the continuous presence of the agonist. Alternatively, the
transient decrease in Vte and Ieq
may be due to activation of transepithelial K+ secretion.
Furthermore, this observation could be caused by more complex
mechanisms; e.g., endogenous PAR-2 activation in native tissues could
contribute to basal and stimulated ion transport, and addition of
trypsin at high concentrations may lead to inactivation of these PAR-2
by cleaveage and/or internalization. However, the trypsin
concentrations (
100 nM) required to induce inhibition of
Vte and Ieq in the plateau phase of
the trypsin response were much higher than the EC50
(21 nM). Therefore, inhibition of ion transport by PAR-2 appears
unlikely under physiological conditions.
Previous studies on cultured renal and pancreatic cells suggested
activation of lumenal Cl and basolateral K+
channels by trypsin along with an increase in intracellular
Ca2+ (2, 26). From the present experiments on
human native colonic mucosa, there is significant evidence for
activation of basolateral Ca2+-dependent K+
channels. However, stimulation of PAR-2 did not activate CFTR or
alternative Ca2+-dependent Cl
channels in
distal colonic tissues. This result confirms previous studies
demonstrating the absence of Ca2+-activated
Cl
channels in human and mouse distal colon (16,
24, 25). Cholinergic stimulation of Cl
secretion
in the mammalian colon has been studied in great detail (6, 17,
24, 25, 32). Here, we compared the effects of carbachol and
trypsin on normal and CF rectal biopsies and found qualitatively
identical responses for both agonists, suggesting that CCH and trypsin
activate similar ion conductances. Moreover, depleting the endoplasmic
reticulum from Ca2+ renders the tissue unresponsive to
trypsin. Thus trypsin is likely to act via release of IP3,
an increase in intracellular Ca2+, and activation of
basolateral Ca2+-dependent K+ channels, which
enhances the driving force for luminal Cl
secretion.
An important aspect of the present results is the reversibility of the
PAR-2 mediated effects. Trypsin cleaves PAR-2 and activates the
receptor irreversibly. Resensitization is due to mobilization of large
Golgi stores and synthesis of new receptors (4). A very
rapid partial recovery was observed in the present study already after
15 min, which is similar to what has been observed in the airways
(7). This finding indicates a very rapid turnover of PAR-2
in native tissue and supports their significance in the regulation of
ion transport in vivo. PAR-2 is also activated by inflammatory
mediators such as tryptase, which is released during mast cell
degranulation. In the human gut, mast cells are resident in the mucosa
associated lymphatic tissue, where they secrete other proinflammatory
cytokines, including tumor necrosis factor- (TNF-
)
(3). TNF-
and interleukin-1 as well as bacterial
lipopolysaccharides have been shown to induce a sustained 10-fold
increase in PAR-2 expression in endothelial cells (28),
supporting participation of PAR-2 in the inflammatory response observed
in chronic inflammatory bowel disease. Interestingly, TNF-
has been
shown to be an essential mediator in inflammatory bowel disease, and
recent clinical trials have shown that treatment with TNF antibody
downregulates inflammation successfully in patients with Crohn's
disease who did not respond to conventional treatment (1,
31). Inflammatory bowel disease, as it occurs in Crohn's
disease, is characterized by mast cell infiltration, which forms an
essential component of intestinal granuloma (23, 29).
Moreover, mast cells have been implicated in affecting ion transport in
the human intestine in a previous study, and changes in ion transport
have been found in patients with inflammatory bowel disease
(9). These results are in parallel with those showing
activation of colonic myocytes by mast cell tryptase and consecutive
disturbances in colonic motility in Crohn's disease (8).
Together with the results from this study demonstrating PAR-2-mediated
activation of ion transport in human distal colon, epithelial PAR-2 may
contribute to the pathophysiology of inflammatory bowel disease and
thus may form a novel pharmacological target.
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ACKNOWLEDGEMENTS |
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We thank the CF patients and volunteers who participated in this study. We gratefully thank Dr. P. Greiner (Children's Hospital, Univ. of Freiburg) for performing rectoscopy procedures and Dr. H. H. Seydewitz (Children's Hospital, Univ. of Freiburg) for genotype analysis of CF patients. We further thank Dr. R. C. Boucher (Cystic Fibrosis Research and Treatment Center, Univ. of North Carolina, Chapel Hill) for discussion and review of the manuscript.
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FOOTNOTES |
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The study was supported by Deutsche Forschungsgemeinschaft KU-1228/1 and KU-756/4-1, Zentrum Klinische Forschung 1 (A2), Univ. of Freiburg, Cystic Fibrosis Australia, Australian Research Council A00104609 and Mukoviszidose E.V.
Present address for M. Mall: Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, The Univ. of North Carolina at Chapel Hill, 7011 Thurston Bowles Bldg., Chapel Hill, NC 27599-7248.
Address for reprint requests and other correspondence: M. Mall, Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, The Univ. of North Carolina at Chapel Hill, 7011 Thurston Bowles Bldg., Chapel Hill, NC 27599-7248 (E-mail: mmall{at}med.unc.edu).
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.
10.1152/ajpgi.00137.2001
Received 29 March 2001; accepted in final form 25 September 2001.
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