Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104
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ABSTRACT |
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Our laboratory demonstrated previously that stimulation of protease-activated receptors (PARs) on the human urothelial carcinoma cell line RT4 results in activation of a calcium-independent phospholipase A2 (iPLA2), leading to arachidonic acid and PGE2 release. In this study, we have examined PAR activation in normal human urothelial cells (HUR) leading to the production of inflammatory or cytoprotective phospholipid metabolites. The presence of both PAR-1 and PAR-2 on HUR was confirmed by immunoblotting. Stimulation of PAR-1 with thrombin or PAR-2 by tryptase leads to activation of a membrane-associated iPLA2 and the production of platelet-activating factor, arachidonic acid, and PGE2. These responses were all blocked by pretreatment with the iPLA2-selective inhibitor bromoenol lactone. Thus stimulation of PAR-1 or PAR-2 on HUR leads to iPLA2-catalyzed phospholipid hydrolysis, resulting in the production of metabolites that may mediate inflammation or provide cytoprotection to the bladder.
thrombin; tryptase; bromoenol lactone
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INTRODUCTION |
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THE PROTEASE-ACTIVATED RECEPTORS (PARs) represent a growing family of G protein-coupled receptors that are activated by certain serine proteases (10). The expression of multiple PARs on a single cell serves to extend the range of proteases to which a cell can respond (20). PARs couple to multiple intracellular signaling pathways related to growth and inflammation, including activation of phospholipases (10) and may have far-reaching implications in diversified cellular responses, particularly in general mechanisms of inflammation and host defense (6, 21). PARs may play important roles in both the anti-inflammatory and proinflammatory behavior of both endothelial and epithelial cells that form the defensive barriers of the body (6). In response to acute cellular activation, epithelial cells release mediators, such as nitric oxide and PGs, that generally have inhibitory effects on inflammatory cells and processes within the mucosa of epithelium-lined organs (11, 14). Thus early activation of epithelial cell PARs may inhibit acute inflammatory responses and provide protection of the organ concerned. In a recent study, we have demonstrated that activation of PARs on RT4 cells, a bladder transitional-cell papilloma, results in activation of iPLA2 and production of arachidonic acid and PGE2 release, which may play a role in cytoprotection during an acute inflammatory reaction (18).
To determine whether the responses we observed in RT4 cells were representative of responses that would be present in normal cells, we subsequently isolated and cultured normal human urothelial cells (HUR) and examined the effects of PAR activation on iPLA2-catalyzed phospholipid hydrolysis. HUR were found to possess both PAR-1 and PAR-2, the activation of which resulted in increased iPLA2 activity and the production of PGE2 and platelet-activating factor (PAF). Because both PAR-1 and PAR-2 activate the same intracellular events, the presence of multiple PARs on HUR probably serves to extend the range of proteases capable of initiating activation of iPLA2, resulting in the production of both inflammatory and cytoprotective phospholipid metabolites.
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MATERIALS AND METHODS |
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Cell isolation and culture. HUR were isolated from adult ureters by using a modified method by Southgate et al. (23) or by growth from explants on the basis of a method described by Trifillis et al. (27). Cells were allowed to grow to confluence in keratinocyte serum-free medium (GIBCO-BRL, Grand Island, NY) supplemented with epidermal growth factor (5 ng/ml; GIBCO-BRL), bovine pituitary extract (50 µg/ml, GIBCO-BRL), cholera toxin (15 ng/ml; Sigma, St. Louis, MO), and 20 U/ml penicillin and 100 µg/ml streptomycin (Sigma). The epithelial origin of the cells was confirmed by the presence of desmosomes identified by electron microscopy and positive staining for epithelial keratins and E-cadherin by using fluoresecent immunocytochemistry.
Stimulation of urothelial cells. Confluent urothelial cells were washed with a HEPES buffer of the following composition (in mmol/l): 133.5 NaCl, 4.8 KCl, 1.2 MgCl2, 1.2 KH2PO4, 10 HEPES, 10 glucose, and 1.2 CaCl2 at pH=7.4. Thrombin (95 U/mg protein; Sigma) and tryptase (1,200 U/mg protein; Promega, Madison, WI) were dissolved in HEPES buffer at a stock concentration 100× the final concentration used. Where appropriate, a stock solution of bromoenol lactone (BEL) in DMSO was diluted with HEPES buffer and added before stimulation by thrombin or tryptase. For measurement of PLA2 activity, the surrounding buffer was removed at the end of the stimulation period and immediately replaced with ice-cold buffer containing (in mmol/l) 250 sucrose, 10 KCl, 10 imidazole, 5 EDTA, and 2 DTT with 10% glycerol, pH = 7.8 (PLA2 assay buffer).
PLA2 activity. HUR suspended in ice-cold PLA2 assay buffer were sonicated on ice six times for 10 s, and the sonicate was centrifuged at 14,000 g for 10 min. The supernatant was centrifuged at 100,000 g for 60 min to separate the membrane fraction (pellet) from the cytosolic fraction (supernatant). PLA2 activity in subcellular fractions was assessed by incubating enzyme with 100 µM [(16:0; 3H, 18:1) or (16:0; 3H, 20:4)] plasmenylcholine or phosphatidylcholine substrates (specific activity of each is 150 dpm/pmol) in assay buffer containing 100 mM Tris, pH = 7.0, and 10% glycerol with either 4 mM EGTA or 1 mM Ca2+ at 37°C for 5 min in a total volume of 200 µl, as described previously (7). Reactions were terminated by the addition of 100 µl butanol, and released radiolabeled fatty acid was isolated by TLC and quantified by liquid scintillation spectrometry.
Arachidonic acid release. Arachidonic acid release was determined by measuring [3H]arachidonic acid released into the surrounding medium from HUR prelabeled with 3 µCi [3H]arachidonic acid (specific activity 100 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA) per culture dish for 18 h. HUR were washed three times with Tyrode solution containing 3.6% bovine serum albumin and incubated at 37°C for 15 min before experimental conditions. At the end of the stimulation period, the surrounding medium was removed to a scintillation vial and represented the amount of radiolabeled arachidonic acid released from HUR. The amount of radiolabeled arachidonic acid in the cells was measured by lysing the cells in 10% SDS. Radioactivity in both surrounding medium and cells was quantified by liquid scintillation spectrometry. Arachidonic acid mobilized from cellular phospholipids was expressed as the percentage of total incorporated radioactivity.
PGE2 release. After stimulation with thrombin or tryptase, the supernatant was removed from HUR and PGE2 was measured with a commercially available immunoassay kit (R&D Systems, Minneapolis, MN).
PAF production. Confluent urothelial cells were washed twice with Hanks' balanced salt solution containing (in mmol/l) 135 NaCl, 0.8 MgSO4, 10 HEPES (pH = 7.4), 1.2 CaCl2, 5.4 KCl, 0.4 KH2PO4, 0.3 Na2HPO4, and 6.6 glucose and incubated with 50 µCi [3H]acetic acid (specific activity 2.5 Ci/mmol, Perkin-Elmer Life Sciences) for 20 min. After stimulation for the selected time interval, lipids were extracted from the cells by the method of Bligh and Dyer (3). The chloroform layer was concentrated by evaporation under N2, applied to a silica gel 60 TLC plate, and developed in chloroform/methanol/acetic acid/water (50:25:8:4 vol/vol). The region corresponding to PAF was scraped, and radioactivity was quantified with liquid scintillation spectrometry. Loss of PAF during extraction and chromatography was corrected by adding a known amount of [14C]PAF as an internal standard. [14C]PAF was synthesized by acetylating the sn-2 position of lyso-PAF with [14C]acetic anhydride using 0.33 M dimethylaminopyridine (DMAP) as a catalyst. The synthesized [14C]PAF was purified by HPLC. The specific activity of the final [14C]PAF product was 300 µCi/µmol.
Electron microscopic determination of desmosomes. Urothelial cell cultures were fixed in 2% glutaraldehyde in PBS followed by postfixation in osmium tetroxide (12). Samples were dehydrated through a graded series of ethanol and embedded in Spurr's resin (12). Ultrathin sections were cut and mounted on copper grids, followed by staining with uranyl acetate plus lead citrate (12). Sections were viewed and photographed on a JEOL 100 CX transmission electron microscope. Micrographs were printed by traditional photographic techniques.
Determination of E-cadherin. Cells were fixed with a solution containing 4% paraformaldehyde, 2% sucrose, 10 mM HEPES, and 10 mM CaCl2 for 5 min at 37°C. Cells were washed several times with PBS and solubilized on ice with 0.5% Triton X-100 and (in mM) 10 PIPES, pH=6.8, 50 NaCl, 300 sucrose, and 3 MgCl2. After washing with PBS containing 1 mM CaCl2 and 1 mM MgCl2, cells were incubated with anti-uvomorulin (1:500 dilution; Sigma) for 1 h, followed by incubation with FITC-conjugated secondary antibody [1:25 dilution anti-rat IgG (whole molecule) FITC conjugate; Sigma] for 1 h. After five washes and mounting in Vectashield medium (Vector Laboratories, Burlingame, CA), cells were viewed in a single plane under a Nikon MRC 1024 laser confocal microscope. Images were captured by using Laser Sharp Version 3.1 software (Bio-Rad, Richmond, CA).
Determination of epithelial keratin.
Epithelial keratin was visualized in HUR by using a modification of the
method of Sun and Green (24) for immunofluorescent staining of keratin fibers; cells were rinsed in PBS with 0.09 mM
CaCl2 and fixed in 3.7% paraformaldehyde, 10 mM HEPES, pH = 7.4, and 1 mM CaCl2 for 10 min, rinsed in PBS, and dipped
in methanol chilled to 20°C for 5 min and then in chilled acetone
for 3 min. After water rinses at 4°C, cells were blocked in PBS
containing 5% normal goat serum and 0.1% albumin and incubated with
anti-epithelial keratin AE1/AE3 mixture (1:200 dilution; ICN
Biochemicals, Aurora, OH) for 1 h at 37°C. After rinses in 0.1%
albumin in PBS, cells were incubated with FITC-conjugated secondary
antibody (1:100 dilution, Sigma) for 1 h. Cells were washed five
times in PBS with 0.1% albumin, rinsed in N-propyl gallate
in PBS (1 mg/ml), and mounted in 0.1 M N-propyl gallate in
90% glycerol and 10% PBS and viewed in a single plane under the laser
confocal microscope. Images were captured by using Laser Sharp Version
3.1 software.
Immunoblot analysis of PARs and PLA2.
HUR were suspended in lysis buffer containing (in mM) 20 HEPES, pH 7.6, 250 sucrose, 2 DTT, 2 EDTA, 2 EGTA, 10 -glycerophosphate, 1 sodium
orthovanadate, and 2 phenylmethylsulfonyl fluoride, as well as (in
µg/ml) 20 leupeptin, 10 aprotinin, and 5 pepstatin A (buffer 2).
Cells were sonicated on ice for six bursts of 10 s and centrifuged
at 14,000 g at 4°C for 10 min to remove cellular debris
and nuclei. Cytosolic and membrane fractions were separated by
centrifuging the supernatant at 100,000 g for 60 min. The
pellet was resuspended in lysis buffer, and the suspension was
centrifuged at 100,000 g for 60 min twice to minimize
contamination of the membrane fraction with cytosolic protein. The
final pellet was resuspended in lysis buffer containing 0.1% Triton
X-100. Protein (cytosol or membrane) was mixed with an equal volume of
SDS sample buffer and heated at 95°C for 5 min before loading onto a
10% polyacrylamide gel. Protein was separated by SDS-PAGE at 200 V for
35 min and electrophoretically transferred to polyvinylidene difluoride
membranes (Bio-Rad) at 100 V for 1 h. Nonspecific sites were
blocked by incubating the membranes with Tris-buffered solution containing 0.05% (vol/vol) Tween-20 [Tris, bicarbonate, sodium, and
Triton X-100 (TBST)] and 5% (wt/vol) nonfat milk for 1 h at room
temperature. The blocked polyvinylidene difluoride membrane was
incubated with primary antibodies to iPLA2 (1:2,000
dilution; Cayman Chemical, Ann Arbor, MI), cPLA2 (1:1,000
dilution: Santa Cruz Biotechnology, Santa Cruz, CA), PAR-1 (1:1,000
dilution: Santa Cruz Biotechnology), or PAR-2 (1:1,000 dilution; Santa
Cruz Biotechnology), for 1 h at room temperature. Unbound
antibodies were removed with three washes with TBST solution, and
membranes were incubated with the appropriate horseradish
peroxidase-conjugated secondary antibodies (1:50,000 dilution,
Amersham, Arlington Heights, IL). After six washes with TBST, regions
of antibody binding were detected with enhanced chemiluminescence
(Pierce Laboratories, Rockford, IL) and exposure to film (Hyperfilm,
Amersham). Multiple exposures of film to the blots were developed, and
exposures that had linear levels of silver grain development were used
for quantitation of band intensity. Immunoblots were quantified with
densitometric analysis (Multi-Analyst, Bio-Rad).
Statistics. Statistical comparison of values was performed by Student's t-test or analysis of variance with a Fisher multiple-comparison test, as appropriate, by using StatMost software (DataMost, Sandy, UT). All results are expressed as means ± SE. Statistical significance was considered to be P < 0.05.
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RESULTS |
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Cells isolated from human ureter were characterized for epithelial
origin on the basis of cell morphology and the presence of
cytokeratin, E-cadherin, and desmosomes (Fig.
1). Cells grew in a cobblestone pattern
comprising polygonal cells containing distinct nuclei (Fig.
1A). Observation by phase microscopy showed cells with
bright intercellular borders (Fig. 1A). Treating fixed cultures with a monoclonal anti-epithelial keratin-AE1/AE3 mixture and
fluorescent-labeled secondary antibody stained positive for these
intermediate filament proteins (Fig. 1B). Immunocytochemical staining for the epithelial adhesion junctional complex molecule E-cadherin showed formation at intercellular junctions (Fig.
1D). In addition, electron microscopic analysis of cultures
of HUR showed the presence of desmosomes, sometimes aligned in tandem, coupling neighboring cells (Fig. 1C).
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Previous studies have demonstrated the presence of PAR-1 and PAR-2 on
epithelial cells (5, 8); however, to date, there are no
data to show the presence of both receptors on HUR. We used immunoblot
analysis to determine that both receptors were present on HUR and found
that both PAR-1 and PAR-2 are present in both the cytosolic and the
membrane fractions (Fig. 2).
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To investigate activation of PLA2 after PAR cleavage, we
used thrombin (which activates PAR-1 but not PAR-2) and tryptase (which
activates PAR-2 but not PAR-1) and measured PLA2 activity. Initial studies determined that the majority of PLA2
activity in HUR was associated with the membrane fraction (Table
1). PLA2 activity measured in
both the cytosolic and the membrane fractions was selective for
arachidonylated phospholipids and was not significantly increased by
the addition of calcium, suggesting that the majority of measured
PLA2 activity was iPLA2 (Table 1). Immunoblot
analysis determined that the majority of iPLA2
immunoprotein was present in the membrane fraction (Fig.
3), with a small amount detected in the
cytosol. Cytosolic PLA2 (cPLA2) immunoprotein
was detected exclusively in the cytosol of HUR (Fig. 3), demonstrating
that both PLA2 isoforms are present. PLA2
activity measured in the cytosol of HUR was not significantly increased
in the presence of Ca2+ (Table 1). Thus cPLA2
appears to contribute minimally to PLA2 activity in HUR,
although the immunoprotein is present.
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After incubation with thrombin (0.05 IU/ml, 2 min), membrane-associated
iPLA2 activity was increased almost fourfold, with no
change in cytosolic iPLA2 activity (Fig.
4). Incubation of HUR with tryptase (2 ng/ml, 2 min) resulted in a twofold increase in membrane-associated
iPLA2 activity. Thus activation of both PAR-1 and PAR-2 on
HUR results in increased membrane-associated iPLA2
activity; however, the magnitude of activation is different for each
receptor. For clarity, PLA2 activity represented in Fig. 4
is that measured in the absence of Ca2+ by using (16:0;
3H, 18:1) plasmenylcholine substrate. However, we have
measured PLA2 activity in the presence and absence of
Ca2+ with all phospholipid substrates to ensure that these
changes are representative of alterations in PLA2 activity
in response to PAR activation and to eliminate the involvement of other
PLA2 isoforms. Increased membrane-associated
iPLA2 activity after PAR activation is likely due to
proteolytic cleavage of the receptors, because membrane-associated
iPLA2 activity was increased significantly after incubation
with the peptide representing the tethered ligand for PAR-1 (SFLLRN,
100 µM, 2 min, iPLA2 activity = 6.9 ± 0.7 nmol · mg protein1 · min
1,
n = 3) and PAR-2 (SLIGKV, 100 µM, 2 min,
iPLA2 activity = 5.0 ± 0.6 nmol/mg protein,
n = 3).
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Pretreatment of HUR with the iPLA2-selective inhibitor BEL
(5 µM, 30 min) (29) significantly inhibited basal
cytosolic and membrane-associated iPLA2 activity and
completely blocked the increases in membrane-associated
iPLA2 activity in response to PAR activation (Fig. 4).
These data further support our hypothesis that the majority of
PLA2 activity in HUR is iPLA2. However, to examine the involvement of cPLA2 in contributing to total
PLA2 activity in HUR, we pretreated HUR with 25 µM methyl
arachidonyl fluorophosphonate (MAFP, an irreversible inhibitor of
cPLA2 and cytosolic iPLA2; see Ref.
1) for 30 min before thrombin or tryptase stimulation.
MAFP had no effect on basal cytosolic (170 ± 35 pmol · mg
protein1 · min
1, n = 4) or membrane-associated (2,278 ± 358 pmol · mg
protein
1 · min
1, n = 4) iPLA2 activity and did not significantly decrease the increase in membrane-associated iPLA2 activity in response
to thrombin (6,335 ± 474 pmol · mg
protein
1 · min
1, n = 4) or tryptase (5,027 ± 442 pmol · mg
protein
1 · min
1, n = 4) stimulation.
To determine whether activation of iPLA2 resulted in the
accumulation of phospholipid metabolites, we measured arachidonic acid
release from HUR after incubation with thrombin and tryptase. Arachidonic acid release was increased significantly after 2-min tryptase stimulation (0.9 ± 0.1 to 1.7 ± 0.2%,
n = 4, P < 0.05), was maximal at 5 min
(3.1 ± 0.3%, n = 4, P < 0.05),
and remained elevated over 20 min. A similar pattern of arachidonic
acid release was observed when HUR were incubated with thrombin, with a
maximal release at 5 min (4.3 ± 0.4%, n = 4, P < 0.05). Released arachidonic acid from HUR was
consequently metabolized to PGE2 (Fig.
5). The release of arachidonic acid (data
not shown) and the production of PGE2 (Fig. 5) were
inhibited by pretreatment of HUR with BEL before PAR activation. To
eliminate a role for cPLA2 in the production of arachidonic
acid and PGE2 in response to thrombin or tryptase stimulation, we pretreated HUR with MAFP for 10 min before PAR activation. Pretreatment with MAFP did not result in significant inhibition of arachidonic acid (data not shown) or PGE2
release (Fig. 5) in response to thrombin or tryptase stimulation of
HUR, further supporting our hypothesis that cPLA2 is not
involved.
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Thrombin and tryptase stimulation of HUR also resulted in a significant
increase in PAF production after 10-min incubation (Fig.
6). Increased PAF production was
inhibited completely by BEL pretreatment (Fig. 6) but was not inhibited
by MAFP (data not shown).
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DISCUSSION |
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Urothelial cells, the transitional epithelial cells that provide the lining from the renal pelvis through the internal urethra, form a defensive barrier that protects the underlying tissue by excluding the penetration of toxins, ions, and bacteria (15). Barrier function of the urothelium can be disrupted by several mechanisms, including inflammation, bacterial infection, toxic chemicals, or mechanical damage, that result in the movement of urinary constituents into the lamina propria and underlying muscle layers. Inflammation of the bladder is the most common clinical disorder in this organ and in most cases is secondary to infection. However, interstitial cystitis (IC) is an inflammatory bladder condition with no known etiology (13, 19, 22). Whether acute or chronic, cystitis is associated with urgency, frequency, and burning, with suprapubic discomfort or pain (19, 22). Inflammation involves both the activation of inflammatory cells and the secondary production of multiple inflammatory mediators, which can act both locally and systemically to enhance and perpetuate the inflammatory response. Activation of PARs on HUR during an inflammatory episode may lead to propagation of the inflammation by means of production of lipid molecules derived from the metabolism of membrane phospholipids.
Thrombin may gain access to the urothelium as a result of plasma exudation through the urothelial cell barrier during inflammation or as a result of mechanical damage (21). During IC, neurogenic inflammation may lead to the release of neuropeptides that activate the secretion of mast cell mediators such as tryptase (2, 25, 26). Thus both thrombin and tryptase may be present in the bladder during inflammation and activate PARs on HUR.
The presence of multiple PARs on the same cell has been postulated previously to expand the range of proteases to which a cell can respond rather than to increase the number of cellular responses to PAR activation (10, 20). In HUR, this also appears to be true, because activation of PAR-1 and PAR-2 results in similar activation of a membrane-associated iPLA2 and subsequent phospholipid hydrolysis. These results are similar to those observed previously in RT4 cells (18), in which activation of PARs resulted in increased iPLA2 activity. Both basal and thrombin- or tryptase-stimulated iPLA2 activities are greater in RT4 cells than in HUR and may be because RT4 is a papilloma cell line. Activation of PAR-1 by thrombin resulted in a greater increase in membrane-associated iPLA2 in HUR than tryptase stimulation of PAR-2. However, the production of both PGE2 and PAF is similar with both agonists, suggesting that cellular responses would be similar and that there is a level of activation of iPLA2 above which no further phospholipid hydrolysis would occur.
PARs are activated by a unique mechanism whereby the NH2 terminus of the receptor is cleaved, leaving a truncated terminus that serves as a tethered ligand (9, 10, 16). Because the PAR cleavage is irreversible, cleaved PARs have been shown to be internalized and degraded by lyosomes and are replaced at the cell surface from an intracellular store. Although this mechanism of inactivation/reactivation has not been demonstrated in HUR, it is possible that such a mechanism exists. In this study, immunoblot analysis demonstrated a significant amount of PARs, and particularly PAR-2, immunoprotein in the cytosolic fraction of HUR. This may represent PARs that were present in an intracellular compartment that were released into the cytosol during subcellular fractionation and would suggest that HUR have a large reserve of PAR-2 compared with PAR-2 expressed on the cell surface. In future studies, we propose to examine the disappearance and reappearance of PARs on the cell surface of HUR after PAR activation.
PLA2 is a key enzyme in the production of lipid mediators from membrane phospholipids. It exists in both secretory and intracellular forms and must be tightly regulated by the cell to prevent autodigestion of the cell membrane (17). When the inflammatory cascade is activated, iPLA2-catalyzed hydrolysis of membrane phospholipids results in the stoichiometric production of free fatty acids and lysophospholipids (17). Arachidonic acid and lysophospholipids can affect membrane function directly and/or serve as the precursors for metabolically active metabolites, such as eicosanoids or PAF. Depending on the balance of production of these metabolites, the net effect on the urothelium may be inflammatory or cytoprotective. In this study, we have demonstrated the concomitant production of PGE2 (a cytoprotective eicosanoid) and PAF (an inflammatory mediator).
Activation of PARs by thrombin or tryptase could mediate inflammation in the bladder by increasing PAF production. PAF is produced by a number of inflammatory cells, including platelets, macrophages, endothelial cells, and epithelial cells, and all appear to possess specific membrane receptors for PAF, functioning in both a paracrine and an autocrine fashion (4). The primary inflammatory actions of PAF include interaction with the production and effect of inflammatory cytokines, contribution to increased expression of other lipid mediators, stimulation of polymorphonuclear neutrophil adherence to the endothelium, and increased vascular permeability (4).
PGE2 has several anti-inflammatory effects, including downregulation of the cytokine response of both macrophages and lymphocytes (4). PGE2 generally has inhibitory effects on inflammation-induced cellular injury within the mucosa of epithelium-lined organs (5, 8). Thus the concomitant production of PGE2 and PAF could lead to both pro- and anti-inflammatory effects. In addition, HUR may produce other eicosanoids in response to PAR activation that may mediate inflammation. However, we did not measure a significant increase in PGI2 or thromboxane A2 production in HUR in response to PAR activation (data not shown).
A recent review (28) has discussed dual roles of protection and inflammation for PAR activation in several tissues. Whether PAR activation would result in primarily a cytoprotective or inflammatory response in the bladder may depend on both the concentration and the time of exposure to the protease and also on the status of the cells. For example, activation of the PARs in HUR in a normal bladder may result primarily in cytoprotection, whereas in a patient with an inflammatory condition such as IC, PAR activation may result in an inflammatory response leading to propagation of inflammation. Recent evidence has shown that PARs are present on neurons (28) and PAR-2 activation causes neuronal sensitization and hyperalgesia. Thus activation of PAR-2 by mast cell tryptase may also contribute to the intense pain associated with IC.
The production of PAF in HUR differs from that in RT4 cells because we did not detect any increase in PAF production in response to PAR stimulation by thrombin or tryptase in RT4 (data not shown). The reason for the difference in PAF production between RT4 and HUR remains unknown at this time. It is possible that the lack of PAF production in RT4 cells could be a result of the immortalization of the cell line, a modification of the cells during multiple passages of cell culture, or a result of these cells being derived from explants of a recurring papillary tumor of the bladder. These data highlight the necessity for the use of normal cells when investigating the response of normal cells to an acute stimulus.
In conclusion, activation of PARs on the surface of HUR results in an increase of iPLA2-catalyzed membrane phospholipid hydrolysis, leading to the production of lipid metabolites that may be pro- or anti-inflammatory.
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ACKNOWLEDGEMENTS |
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The authors thank Pamela Kell for technical assistance and Dr. David Lagunoff for assistance with the confocal microscopy studies.
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FOOTNOTES |
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This work was supported in part by the Interstitial Cystitis Association (A. Rickard), the Roland Quest Trust Fund of the St. Louis Community Foundation (A. Rickard), and the American Heart Association, Heartland Affiliate (J. McHowat).
Address for reprint requests and other correspondence: J. McHowat, Dept. of Pathology, St. Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: mchowatj{at}slucare1.sluh.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.
July 9, 2002;10.1152/ajprenal.00072.2002
Received 19 February 2002; accepted in final form 30 June 2002.
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