Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612-7343
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ABSTRACT |
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We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+]i) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+]i increase in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but preexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40-60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 3 h later. Loss of cell retractility was coupled to decreased cell surface PAR-1 expression as determined by immunofluorescence. Cell surface PAR-1 disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-insensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.
proteolytically activated thrombin receptor; human pulmonary artery endothelial cells; endothelial monolayer resistance; cytosolic calcium concentration
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INTRODUCTION |
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ACTIVATION OF ENDOTHELIAL cells by thrombin results in cell retraction, stimulation of prostacyclin synthesis, increased cytosolic Ca2+ concentration ([Ca2+]i), expression of cell surface P-selectin and intercellular adhesion molecule-1, and increased transendothelial permeability to albumin (15). Studies have shown that proteolytically activated thrombin receptor (PAR-1) is expressed on vascular endothelial cell membranes (18). We and others have shown that thrombin-induced activation of PAR-1 in endothelial cells increases endothelial permeability (6, 7, 14), a response that results from retraction of adjacent endothelial cells, which increases the dimension of the paracellular pathway (13, 25).
The diverse effects of thrombin in endothelial cells are activated by thrombin binding to the G protein-coupled seven-transmembrane PAR-1 (3, 27). Thrombin binds to the hirudin-like domain in the extracellular amino-terminal region of PAR-1 (27) and cleaves the receptor between Arg-41 and Ser-42 (27). The newly formed amino-terminal region binds to the second extracellular loop and amino-terminal extension regions on the PAR-1 to transmit signals (7, 17, 19). Synthetic peptides corresponding to the new amino terminus are capable of activating the receptor (27). Exposure to thrombin causes rapid desensitization such that subsequent thrombin challenge produces little or no Ca2+ signaling (24). Proteolysis of PAR-1 by thrombin irreversibly cleaves the receptor, a phenomenon distinct from activation of other G protein-coupled receptors (8, 27). The internalization of the activated PAR-1 is, in part, the result of phosphorylation of the carboxy terminus of the receptor by G protein-linked receptor kinases (8, 11, 21).
The regulation of PAR-1 resensitization in endothelial cells after the internalization of the degraded receptor is not well understood. Brass and colleagues (2, 10) showed that activation of PAR-1 by thrombin in megakaryoblastic HEL and CHRF-288 cell lines (which do not have an intracellular pool of PAR-1) resulted in rapid internalization of the receptor and >90% degradation in acidic lysosomes. Horvat and Palade (9) showed by immunoelectron microscopy of human umbilical vein endothelial cells (HUVEC) the presence of preformed PAR-1 stores in intracellular vesicular compartments. Hein et al. (8) showed that this intracellularly stored PAR-1 pool (which they localized in the Golgi apparatus) could translocate to the HUVEC surface within 60 min of thrombin exposure, suggesting that endothelial cells could be rapidly reactivated by thrombin. In the present study, we investigated the kinetics of endothelial cell surface PAR-1 reexpression and the "activation potential" of the cell surface PAR-1 upon its reexpression. The results indicate that the endothelial cell surface PAR-1 can be rapidly restored even in the presence of continuous exposure to a low concentration of thrombin, but that cellular PAR-1 pools are depleted by high concentrations of thrombin and longer exposure times. We provide evidence of a direct relationship between cell surface PAR-1 expression and activation of Ca2+ signal and endothelial cell retraction.
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MATERIALS AND METHODS |
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Materials.
Human -thrombin was obtained from Enzyme Research Laboratories
(South Bend, IN). Hanks' balanced salt solution (HBSS) and trypsin
were purchased from GIBCO (Grand Island, NY). Fetal bovine serum (FBS)
was purchased from HyClone (Logan, UT). Human pulmonary artery
endothelial cells (HPAEC) and EBM-2 medium were from Clonetics (San
Diego, CA). Electrodes for endothelial monolayer resistance measurements were purchased from Applied Biophysics (Troy, NY). Fura
2-AM (cell permeant) was purchased from Molecular Probes (Eugene, OR).
Cycloheximide was purchased from United States Biochemical (Cleveland,
OH). Anti-PAR-1 monoclonal antibody (MAb) raised against amino-terminal
extension sequence Trp-Glu-Asp-Glu (recognizing both naive
and thrombin-cleaved PAR-1) was obtained from Immunotech (Westbrook, ME).
Endothelial cell culture. HPAEC were grown in EBM-2 medium supplemented with 10% FBS. Cells were cultured on tissue culture dishes coated with 0.1% gelatin and were used between passages 4 and 8.
[Ca2+]i measurement. Thrombin-induced increase in [Ca2+]i was measured using the Ca2+-sensitive fluorescent dye fura 2 (24, 30). Cells were grown to confluence on gelatin-coated glass coverslips and then washed two times with serum-free medium and incubated for 2 h at 37°C in culture medium containing 1% FBS. After thrombin exposure, cells were washed once and loaded with 2 µM fura 2-AM for 1 h at 25°C. After the loading, cells were washed with HBSS and placed in a Sykes-Moore perfusion chamber positioned on the stage of a Nikon Diaphot microscope, which was coupled to a Deltascan microspectroflourometric system (Photon Technology International, Princeton, NJ). An optically isolated field of endothelial cells was excited alternately with wavelengths of 340 and 380 nm, and emitted light was collected at 510 nm with a photomultiplier. Background autofluorescence (in the absence of fura 2) determined at the beginning of each day of experiments was subtracted automatically during data collection. At the end of each experiment, 10 µM ionomycin was added to obtain the fluorescence of Ca2+-saturated fura 2 and 0.1 M EGTA was added to obtain the fluorescence of free fura 2. The ratios of fluorescence at 340 nm to that at 380 nm and the values for [Ca2+]i were calculated using PTI software.
Cell surface PAR-1 immunofluorescence. HPAEC were grown to confluence on glass coverslips. The cells were washed three times with serum-free medium and kept for 2 h at 37°C in medium containing 1% FBS before agonist treatment. After thrombin treatment, cells were washed three times with HBSS and fixed with 1% paraformaldehyde for 15 min at 22°C. Cells were then blocked with 5% BSA at 4°C for 60 min before addition of anti-PAR-1 MAb (5 µg/ml) for 60 min at 4°C. The anti-PAR-1 MAb binding was detected by incubating rhodamine-labeled goat anti-mouse IgG (5 µg/ml) for 30 min at 4°C. Cell surface fluorescence was visualized using digital imaging fluorescence microscopy (Diaphot 200, Nikon Instruments, Fair Lawn, NJ). Specimens were viewed with a ×100 objective. Excitation and emission wavelengths were selected with a filter set optimal for the fluorescence of tetramethylrhodamine. Images were collected for analysis with a cooled charge-coupled device array detector (Imagepoint, Photometrics, Tucson, AZ) with a spatial resolution of 768 vertical by 452 horizontal. Images were acquired by the detector with a 6-s integration period and then digitized at 8 bits/pixel brightness resolution and transferred directly to a personal computer frame memory.
Endothelial cell shape change.
The time course of endothelial cell shape change (in real time) in
response to thrombin was measured according to procedures described by
us (25). In brief, HPAEC were grown to confluence on small gold
electrodes (4.9 × 104
cm2). Before experiments,
monolayers were washed two times with serum-free medium and incubated
for 2 h in 1% serum-supplemented culture medium. The small electrode
and the large electrode were connected to a phase-sensitive lock-in
amplifier. A constant current of 1 µA was applied by a 1-V, 4,000-Hz
alternating current connected serially to a 1-M
resistor between the small electrode and the larger counter electrode.
The voltage change between the small electrode and the larger counter
electrode was continuously monitored by the lock-in amplifier, stored,
and processed on a computer. The data are presented as the change in
the resistive (in-phase) portion of the impedance normalized to its
initial value at time zero as described (25, 26).
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RESULTS |
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Desensitization of PAR-1 and its recovery in endothelial cells. Endothelial cells were pretreated with 10 nM (1.0 National Institutes of Health unit) or 25 nM thrombin for different times before measurement of the increases in [Ca2+]i. Cells were challenged for 30, 60, 90, or 120 min (as shown in Fig. 1, A and B) with 10 or 25 nM thrombin, washed with DMEM, and incubated in thrombin-free medium before the second thrombin challenge at 3 h. Pretreatment of HPAEC with either 10 or 25 nM thrombin for 30 min resulted in the same increases in the [Ca2+]i at 3 h as in control cells (Fig. 1, A and B). Cells pretreated with 10 nM thrombin for 60, 90, or 120 min remained responsive to thrombin at 3 h (Fig. 1A). In contrast, cells preexposed to 25 nM thrombin for 60, 90, or 120 min did not respond to thrombin stimulation at 3 h (Fig. 1B). Thus 25 nM thrombin exposure for >30 min fully desensitized PAR-1 in HPAEC, indicating that both concentration and duration of thrombin exposure are critical determinants of PAR-1-activated Ca2+ signaling.
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Loss of cell surface PAR-1 and its recovery within 60-90 min. We determined the surface expression of PAR-1 by immunofluorescence after exposing the cells to thrombin for different times. After thrombin exposure, cells were fixed, nonspecific binding was blocked, and cells were incubated with anti-PAR-1 MAb (see MATERIALS AND METHODS). Both 10 and 25 nM thrombin exposure for 30 min at 37°C prevented cell surface antibody (Ab) staining compared with control cells (Fig. 2), indicating the rapid loss of cell surface PAR-1 with both thrombin concentrations. Cells exposed to 25 nM thrombin for 60 min also showed no Ab staining (Fig. 2); however, cell surface Ab staining reappeared within this time in cells exposed to 10 nM thrombin (Fig. 2). These differences between the two thrombin concentrations were clearly evident when the thrombin pretreatment period was extended to 90 min in that cell surface PAR-1 was present only in cells exposed to 10 nM for 90 min (Fig. 2).
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Endothelial cells become refractory to thrombin with loss of cell surface PAR-1. We measured endothelial cell retraction response in real time by determining the effects of thrombin on HPAEC monolayer resistance (10). Addition of 10 or 25 nM thrombin caused a 40-60% decrease in resistance, whereas DMEM had no effect (Fig. 4), indicating that activation of PAR-1 in HPAEC caused the rapid retraction of confluent endothelial cells. The decreased endothelial monolayer resistance returned to the normal range within 2 h. Thrombin concentrations within the range 0.1-100 nM produced similar results (data not shown). Endothelial cell retraction was not inhibited by cycloheximide (6 µg/ml) pretreatment (data not shown), indicating that it was protein synthesis independent.
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Long-term recovery of PAR-1 activity occurs at 18 h after PAR-1 depletion. Because continuous exposure of 25 nM thrombin for 3 h depleted PAR-1 (Fig. 2) and resulted in HPAEC becoming refractory to thrombin (Fig. 4, A and B), we determined the reversibility of this process. Cells were cultured on electrodes as described above to measure endothelial cell monolayer resistance. HPAEC were first treated with 10 or 25 nM thrombin, after which the cells were challenged with thrombin at different time intervals. After the initial 25 nM thrombin treatment, cell function was restored at 18 h after thrombin in the absence of cycloheximide (Fig. 5A). Similar results were obtained when cells were challenged with 10 nM thrombin twice at 0 and 3 h to deplete PAR-1 (data not shown). Although the endothelial cell retractile response was restored at 18 h after the initial thrombin challenge, the response did not recover following treatment with cycloheximide (Fig. 5A). Cells exposed to 25 nM thrombin for 3 h demonstrated cell surface PAR-1 expression at 18 h, whereas pretreatment of these cells with cycloheximide prevented cell surface PAR-1 expression at 18 h (Fig. 5B).
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DISCUSSION |
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G protein-coupled receptors undergo homologous desensitization after
short-term exposure to their agonists (1, 5). Desensitization involves
a combination of sequential events: receptor phosphorylation, internalization, intracellular degradation, dephosphorylation, and
recycling of internalized receptors to the cell surface (1, 5). This
process has been well studied for the
2-adrenergic receptors
(
2-AR) (1). However, PAR-1, a G
protein-coupled receptor, is not recycled to the cell surface after its
internalization because it is irreversibly modified by proteolysis (1,
27). PAR-1 is also specialized because of a mobilizable pool of the receptor in endothelial cytosol (8, 9). In the present study, we
determined the characteristics of cell surface PAR-1 disappearance and
its reappearance in endothelial cells. Because PAR-1 activation in
vascular endothelial cells causes cell retraction, a major factor in
the development of increased endothelial permeability via the
paracellular pathway (15), we also determined the relationship of cell
surface PAR-1 expression to activation of thrombin-induced Ca2+ signaling and retraction of
endothelial cells.
Brass and colleagues (2, 10) investigated thrombin-dependent
desensitization of PAR-1 in the megakaryoblastic HEL and CHRF-288 cell
lines using Abs directed against the tethered-ligand domain and
thrombin-binding (hirudin-like domain) region of PAR-1. Exposure to
10-20 nM thrombin for 10 min caused internalization of >85%
receptors, and the receptors were subsequently degraded in acidic
lysosomes (2, 10). However, some cleaved receptors (i.e., the
"activated" receptors) were recycled to the cell surface but
could not be activated by thrombin. Brass and colleagues (2, 10) showed
that these megakaryoblastic cell lines (which exhibit platelet
properties) responded to thrombin 22 h after the initial thrombin
challenge and that this response was dependent on de novo protein
synthesis. Hein et al. (8) expressed FLAG epitope-tagged PAR-1 and 2-AR in Rat1
fibroblasts and followed intracellular receptor traffic after exposure
to agonists. After thrombin cleavage, the removal of epitope from
receptors allowed the naive receptors to be distinguished from cleaved
PAR-1 (8). In control cells, PAR-1 was localized on the cell surface
and intracellular compartments; however,
2-AR was localized only on the
cell surface. They concluded that PAR-1 (unlike
2-AR) is stored in a preformed
state in the cytosol (8), like the
N-formyl peptide receptor in
neutrophils (20). Moreover, stimulation of Rat1 fibroblasts expressing
both PAR-1 and
2-AR resulted in
rapid internalization of both receptors into endosomal vesicles (8).
The internalized
2-AR was
recycled to the cell surface, whereas PAR-1 was targeted to lysosomes
(8). Thrombin-challenged Rat1 fibroblasts also did not show an increase in
[Ca2+]i
when challenged with thrombin within 5 min after the initial thrombin
exposure; however, cells responded to the second thrombin challenge at
60 min with a normal increase in
[Ca2+]i,
a response that was insensitive to cycloheximide (8). These results
indicated that Rat1 fibroblasts have an intracellular pool of PAR-1
that upon activation can signal PAR-1 translocation to cell surface. An
intracellular PAR-1 pool has also been observed in HUVEC (8, 9). In
contrast, the PAR-1-expressing megakaryoblastic cell lines lacked an
intracellular PAR-1 pool (2, 10).
In the present study, we determined the characteristics of PAR-1 cell surface expression using HPAEC to address the "turning off" and reactivation of PAR-1 signaling. We determined cell surface PAR-1 expression using an anti-PAR-1 MAb that recognized both the cleaved and naive cell surface PAR-1 (8, 27). The results indicate that the concentration of thrombin and exposure time are both critical determinants of loss of cell surface PAR-1. The higher concentration of thrombin (25 nM) maintained for 1-2 h depleted the cell surface PAR-1 such that the cells did not respond to thrombin for up to 18 h. The lower concentration of thrombin [10 nM, in the range of 0.1-10 nM expected after conversion of prothrombin to thrombin (12, 22)] also produced the loss of cell surface PAR-1 and Ca2+ signaling, but this response was reversible within 60-90 min after the initial thrombin challenge. These results indicate that cleavage of PAR-1 and its internalization abrogate the signaling; however, early recovery of cell surface PAR-1 can occur after short-term exposure to thrombin of ~30 min. These findings support the hypothesis that the cytosolic pool of PAR-1, which can be rapidly translocated to the cell surface (8, 28), serves an important function in enabling the endothelial cell to rapidly restore its responsiveness to thrombin after receptor inactivation by cleavage and internalization.
Exposure to 25 nM thrombin for 30 min resulted in an immediate loss of the cell surface PAR-1, which was followed by the return of PAR-1 within 90 min after thrombin treatment. These results raise the possibility that depletion of the cell surface PAR-1 itself signals translocation of cytosolic stores. Internalization of receptors and their uncoupling from heterotrimeric G proteins may signal the mobilization of the receptor to the cell surface (11). Another possibility is that the thrombin can signal translocation of cytosolic PAR-1 to the cell surface by activation of second messenger pathways (which have not been thus far defined) (8, 28).
We observed that loss of cell surface PAR-1 and Ca2+ signaling were coupled to the inactivation of the endothelial cell retraction response. We measured transendothelial electrical resistance to study retraction of endothelial cells following thrombin exposure, as this is a critical event responsible for increasing endothelial permeability (13, 25). The cells became refractory to the second stimulus after having been exposed to thrombin to deplete the cell surface PAR-1. We showed that the loss of cell surface PAR-1 and Ca2+ signaling abrogated the thrombin-induced retraction of endothelial cells. This finding is in accord with the important function of Ca2+ in signaling the thrombin-induced retraction of endothelial cells by activation of the myosin light chain kinase-dependent actin-myosin contractile machinery of endothelial cells (16, 23, 29).
Although the cell surface PAR-1 could be depleted in endothelial cells as the result of thrombin exposure and thus desensitize the receptor to further thrombin stimulation, we observed that the cells remained responsive to histamine. Histamine produced a sharp drop in electrical resistance characteristic of cell retraction (16) in cells that were not responsive to thrombin. We conclude from this observation that thrombin exposure of endothelial cells produced homologous desensitization of PAR-1.
Exposure of endothelial cells to 25 nM thrombin produced a long-lived desensitization lasting ~18 h, at which point thrombin exposure produced its characteristic decrease in electrical resistance. This response was sensitive to cycloheximide, suggesting that it required new synthesis of PAR-1. Because we have shown that thrombin can signal the activation of PAR-1 mRNA, a process regulated by the activation of tyrosine kinase pathways (4), the delayed resensitization after loss of cell surface PAR-1 appears to be distinct in endothelial cells vs. platelets because it involves de novo synthesis of PAR-1.
We observed that challenge of endothelial cells at 18 h resulted in a sharp characteristic drop in electrical resistance, suggesting that the cell surface PAR-1 had been restored. Interestingly, subsequent challenge with 10 nM thrombin, 3 h later, which normally would have elicited another decrease in resistance because of translocation of the cell surface PAR-1 to the membrane, failed to produce such an effect. This finding raises the possibility that cell surface PAR-1 is preferentially replenished at the expense of the cytosolic pool following new synthesis of PAR-1. Whether preferential targeting of the newly synthesized PAR-1 to the membrane is responsible for this effect will need to be explored.
In summary, we have shown that thrombin concentration and its exposure time regulate endothelial cell surface PAR-1 expression. Rapid resensitization occurs by replenishment of the cell surface PAR-1 by the translocation of preformed cytosolic PAR-1 to the membrane within 60-90 min of depletion of the surface PAR-1. In contrast, the delayed process of resensitization involves new PAR-1 synthesis. The finding that thrombin modulates the rapid and delayed surface expression of PAR-1 in endothelial cells suggests a novel means of preventing the increase in endothelial permeability involving the desensitization of PAR-1.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-27016, HL-45638, and T32-HL-07829.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: C. Tiruppathi, Dept. of Pharmacology (M/C 868), University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612-7343.
Received 20 July 1998; accepted in final form 9 September 1998.
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