(Circulation. 1999;99:2590-2597.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Protease-Activated Receptor-2 Involvement in Hypotension in Normal and Endotoxemic Rats In Vivo
From the Dipartimento di Farmacologia Sperimentale, Naples, Italy (C.C., M.B., R.S., G.C.); Dipartimento di Scienze Farmaceutiche, Penta (Sa), Italy (A.P.); the School of Biology and Biochemistry, Queen's University of Belfast, Medical Biology Centre, Belfast, UK (B.W., P.H.); and Clinical Sciences Research Centre, St Bartholomew's, and the Royal London School of Medicine and Dentistry, London, UK (A.C., S.K., G.L.H.).
Correspondence to Professor Giuseppe Cirino, PhD, Dipartimento di Farmacologia Sperimentale, Via Domenico Montesano 49, 80131 Naples, Italy. E-mail cirino{at}cds.unina.it
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Abstract |
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Background—The protease-activated receptor-2 (PAR-2) is expressed by vascular endothelial cells and upregulated by lipopolysaccharide (LPS) in vitro. PAR-2 is activated by a tethered ligand created after proteolytic cleavage by trypsin or experimentally by a synthetic agonist peptide (PAR-2AP) corresponding to the new amino terminus of the tethered ligand.
Methods and Results—Intravenous administration of PAR-2AP (0.1, 0.3, and 1 mg/kg) to rats caused a dose-dependent hypotension. A scrambled peptide was without effect. A specific trypsin inhibitor, biotin–SGKR-chloromethylketone, inhibited trypsin-induced hypotension but not that stimulated by PAR-2AP. In animals treated with LPS 20 hours earlier, we found an increased sensitivity to trypsin and PAR-2AP in the hypotensive response. In particular, PAR-2AP caused hypotension at a low concentration of 30 ng/kg. Moreover, PAR-2 was immunolocalized to endothelial and smooth muscle cells in aorta and jugular vein in LPS-treated rats, and increased levels of PAR-2 mRNA were shown by reverse transcription–polymerase chain reaction analysis.
Conclusions—Our findings suggest that PAR-2 is important in the regulation of blood pressure in vivo. A functional upregulation of PAR-2 by LPS was demonstrated by the activity of concentrations of PAR-2AP that were inactive in normal animals. We conclude that PAR-2 may play an important role in the hypotension associated with endotoxic shock and may represent a new therapeutic target.
Key Words: receptors • trypsin • endotoxemia • hypotension • shock
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Endothelial cells represent a major interface with plasma proteins and play a key role in modulating platelet adhesion, aggregation, and fibrinolysis. The vascular tone is regulated mainly locally by the release of vasoconstrictory or vasodilatory factors, such as nitric oxide, endothelin, and eicosanoids, particularly prostacyclin and thromboxane. In the late 1930s, Rocha e Silva1 2 3 made the observation that an intravenous injection of trypsin caused hypotension. Trypsin also caused histamine release from perfused guinea pig lungs4 and other tissues, including liver5 and blood leukocytes.6
These early observations may be relevant to the recent finding that at
the molecular level, trypsin is able to activate the
protease-activated receptor-2 (PAR-2). PAR-2 is a G
protein–coupled receptor that, like the thrombin receptor PAR-1, is
activated by proteolytic cleavage of its extracellular
amino-terminus domain to create a new N-terminus that functions as a
tethered ligand.7 8 PAR-2 expressed in Xenopus
oocytes can be activated by low concentrations of trypsin (0.3
to 3 nmol/L) or by a short synthetic agonist peptide (PAR-2AP)
corresponding to the new amino terminus exposed after trypsin cleavage,
and the agonist peptide (SLIGRL-NH2) has an
EC50 of 
5 µmol/L.7 8
Recent immunolocalization studies show that PAR-2 is expressed in a
variety of normal human tissues, but its
physiological roles are unknown.9 10
Significantly, in contrast to PAR-1, PAR-2 is upregulated 5- to 10-fold
in endothelial cells in vitro after exposure to
lipopolysaccharide (LPS), interleukin-1, and tumor necrosis
factor-
, suggesting a possible role for PAR-2 in
endotoxemia.11 Here, we have investigated the effect on
blood pressure of trypsin and human PAR-2AP on normal and LPS-treated
rats.
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Methods |
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Reagents
PAR-2–activating peptide (PAR-2AP) from human (SLIGKV-NH2) and control peptide with a scrambled sequence (KGLVS-NH2) were synthesized by standard solid-phase 9-fluorenylmethoxycarbonyl (FMOC) chemistry with an automated peptide synthesizer (Applied Biosystems, model 432A). Peptides were purified by reverse-phase high-performance liquid chromatography, and their identity was confirmed by mass spectroscopy as described previously.12 Polymyxin B, mepyramine, cyproheptadine, and nordihydroguaiaretic acid (NDGA) were from Sigma Chemical Co. Activated CH-Sepharose 4B was from Pharmacia-LKB Biotechnology. Trypsin (porcine pancreas, EC 3.4.21.4, 13 000 to 20 000 BAEE U/mg), indomethacin, atropine, bradykinin, nitro-L-arginine methyl ester (L-NAME), LPS from Escherichia coli (serotype 0127:B8), salts, and buffers were purchased from Sigma. Glyceryltrinitrate (GTN) was a generous gift of Dr Vincenzo Greco (Cardiology Unit, Naples, Italy). The ETA/ETB endothelin inhibitor (SB209670) was obtained from SmithKline Beecham, and HOE-140 was obtained from Hoechst. Hirulog was a generous gift of Dr Maraganore (Biogen, USA). The biotinylated inhibitor biotin-Ser-Lys-Gly-Arg-CH2CP, which is based on the P1-P4 residues of the PAR-2 cleavage site, was synthesized according to the method of Kay et al.13
Blood Pressure Measurement
Rats (Charles River, Wilmington, Mass, 250 to 300 g)
were anesthetized with urethane (solution 15% wt/vol;
1.5 g/kg IP), and the left carotid artery and right jugular vein were
cannulated for blood pressure measurement and drug administration,
respectively. All the drugs tested were administered
intravenously as a slow bolus injection except L-NAME,
which was infused through the tail vein with a pump (Harvard, model 21)
at 3 µg · kg-1 ·
min-1 for 45 minutes. This dose was previously
determined not to cause an increase in blood pressure but to reduce
acetylcholine-induced hypotension. PAR-2AP (0.1, 0.3, and 1 mg/kg) or
trypsin (0.2, 0.6, and 2 mg/kg) was administered through the jugular
vein every 20 minutes 3 times consecutively. Blood pressure values were
expressed as mean arterial blood pressure (MABP).
Studies in LPS-Treated Rats
Rats (Charles River, 250 to 300 g) were treated with LPS
(13.5x106 U/kg IV) or saline. After 6, 12, or 20
hours, animals were anesthetized and blood vessels cannulated
as described above for blood pressure measurement and drug
administration. PAR-2AP (0.03, 0.3, and 1 mg/kg) or trypsin (0.02, 0.2,
0.6, and 2 mg/kg) was administered through the jugular vein. In some
cases, rats were killed and the abdominal aorta and jugular vein were
removed, immediately frozen in liquid nitrogen, and stored at -70°C
for immunohistochemistry or reverse transcription–polymerase chain
reaction (RT-PCR) studies.
In Vitro Studies
Rats that had received saline or LPS were killed, and the
thoracic aorta was removed and immersed in cold gassed (95%
O2/5% CO2) Krebs solution
composed of (in nmol/L) NaCl 115.3, KCl 4.9,
CaCl2 1.46, MgSO4 1.2,
KH2PO4 1.2,
NaHCO3 25.0, and glucose 11.1. Aortas were
cleaned of adherent connective tissue and cut into rings (5 mm
long) and placed in 2 mL isolated organ bath filled with gassed Krebs
solution maintained at 37°C. The rings were connected to an isometric
transducer (model 7004, Basile), and changes in tension were
recorded continuously with a polygraph linear recorder (WR 3310
Graphtec). The rings were washed at 30-minute intervals during the
equilibration period, and a tension of 0.5 g (basal tone) was
applied. For each ring mounted, the molar concentration that produced
80% of the maximum contraction (EC80) was
established. Briefly, arteries were contracted with cumulative
concentrations of phenylephrine (PE, 0.01 to 3
µmol/L), and the EC80 was determined from the
curve obtained before the rings were washed with Krebs solution. Aortas
were then contracted with PE EC80, and when a
stable tone was reached, a single concentration of acetylcholine
(1 µmol/L) was applied to assess the endothelial
response. Rings that showed relaxation of <70% were discarded. On the
subsequent stable PE contraction, the effect of PAR-2AP or control
peptide (10, 30, and 100 µmol/L) was assessed. Peptides were
also tested in the presence of L-NAME (100 µmol/L). Data are
expressed as percent of relaxation calculated as percent of the maximum
relaxation induced by acetylcholine in the same tissue.
Preparation of PAR-2 Antibodies
A peptide corresponding to the activation site of human PAR-2
(SKGRSLIGKVDGTSHVTGK-NH2, residues 33 to 51) was
synthesized as a multiple antigenic construct and was used as an
immunogen.14 Rabbit anti–PAR-2 IgGs were purified with an
affinity column of the linear peptide coupled to activated
CH-Sepharose 4B, as previously described.15 Preimmune sera
were purified with a protein-A Sepharose column to yield control
IgG.
Immunohistochemistry
Aorta and jugular veins removed from rats were stored at
-70°C before use. Serial cryostat (Bright) sections (6 µm)
were cut and mounted on APES-coated slides (Sigma) and dried
overnight at room temperature. Sections were fixed in cold acetone for
1 minute before blocking with 10% FCS in 0.2 mol/L Tris-HCl buffer for
15 minutes to reduce nonspecific staining. Anti–factor VIII rabbit IgG
(Dako), affinity-purified rabbit anti–PAR-2 IgG, or preimmune control
IgG was diluted in 2% FCS Tris-HCl (1, 5, 10, and 20 µg/mL), applied
to sections, and incubated overnight in a humid chamber at 4°C. After
a washing step with PBS, sections were dried and incubated with
biotinylated anti-rabbit IgG (Dako) for 30 minutes, followed by
avidin–biotinylated alkaline phosphatase complex (Dako) incubated for
a further 30 minutes at room temperature. Color was developed with
Vector Red (Vector Laboratories) containing 1 mmol/L levamisole to
block endogenous alkaline phosphatase activity. The
sections were counterstained with hematoxylin, mounted, and examined
with a light microscope (Photomat FX4, Nikon).
RT-PCR Analysis
Aortas and jugular veins were removed from control and
LPS-treated rats, snap-frozen in liquid nitrogen, and stored at
-70°C. Total RNA was isolated with RNAzol solution (Biogenesis)
according to the manufacturer's instructions. RNA purity was estimated
by measurement of optical density at 260/280 nm. Total RNA (5 µg) was
subjected to first-strand cDNA synthesis in a 10-µL reaction
containing 250 mmol/L Tris-HCl (pH 8.3 at 20°C), 375 mmol/L
KCl, 15 mmol/L MgCl2, 1 mmol/L DTT,
1 mmol/L of each dNTP, and 20 U RNase inhibitor, in
the presence of 1.5 µg oligo dT(12-18) primer
and 200 U Superscriptase (all from Life Technologies). After completion
of first-strand cDNA synthesis, the reaction was stopped by heat
inactivation (5 minutes, 95°C) and diluted to 50 ng/µL RNA
equivalents with water. cDNA amounts equivalent to 100 ng of total RNA
were subjected to PCR in a 50-µL reaction volume containing 10
mmol/L Tris-HCl (pH 9 at 25°C), 50 mol/L KCl, 1.5 mmol/L
MgCl2, 0.01% (wt/vol) gelatin, 0.1% (vol/vol)
Triton X-100, 2 mmol/L DTT, 200 µmol/L of each dNTP, 1
µmol/L of each primer, and 0.2 U of Taq DNA polymerase (AB
Biotechnology) under the following conditions: denaturation for 30
seconds at 94°C, primer annealing for 1 minute at 58°C, and primer
extension for 1 minute at 72°C. The PCR products (10 µL) were
electrophoresed through 1% agarose gels and viewed by UV illumination.
Primers used were 5'-ATGCGAAGTCTCAGCCTG-3' (sense) and
5'-TCAGTAGGAGGTTTTCCG-3' (antisense) to amplify a 1900-bp PCR
product for rat PAR-2, and 5'-ACCACAGTCCATGCCATCAC-3' (sense)
and 5'-TCCACCACCCTGTTGCTGTA-3' (antisense) to amplify a 400-bp
product for GAPDH.
Statistics
Data were analyzed by ANOVA, 1- or 2-way as appropriate,
followed by Dunnett's test.
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Results |
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Effect of PAR-2AP and Trypsin on MABP
As shown in Figure 1A
, intravenous administration of human PAR-2AP (0.1, 0.3, and
1 mg/kg IV) caused a dose-related hypotension. The change in MABP was
characterized by a rapid fall lasting 30 seconds for 0.1 mg/kg, 1
minute for 0.3 mg/kg, and 2 minutes for 1 mg/kg (Figure 2A). Trypsin-induced hypotension (Figure 1B) was very similar to that caused by PAR-2AP. The kinetics of
the hypotension and the return to basal blood pressure levels for the
doses of 0.2 and 0.6 mg/kg occurred at almost the same time intervals
as seen with PAR-2AP (Figure 2B). A dose of 2 mg/kg took 3
minutes to return to the basal value. There was no tolerance to the
hypotensive effect of both PAR-2AP and trypsin (data not shown).
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Effect of Inhibitors on Hypotension
Atropine (0.5 mg/kg), HOE-140 (0.1 mg/kg), NDGA (15 mg/kg),
and mepyramine (5 mg/kg IV) had no effect on the hypotension caused by
trypsin (data not shown). The doses chosen are able to cause at least
50% inhibition of the response to the specific agonist. However,
indomethacin (5 mg/kg IV) administered 1 hour before
trypsin (2 mg/kg) caused a reduced hypotensive response (Figure 3A), from 47±2.4 to 16±2.2 mm Hg
(n=4; P<0.01). The mixed endothelin
(ETA/ETB)
antagonist SB209670, at a dose of 1 mg/kg IV given 30
minutes before trypsin (2 mg/kg) administration, caused a reduction of
hypotensive response (Figure 3B) from 38±7 to 18±3.4
mm Hg (n=4; P<<0.01). L-NAME was given as an infusion at a
rate 3 µg · kg-1 ·
min-1. This dose was determined, in a separate
set of experiments, not to cause hypertension (data not shown). Trypsin
(2 mg/kg IV)–induced hypotension before the L-NAME infusion was
started was 46±8.2 mm Hg. After L-NAME, the hypotension was
29.3±10.4 mm Hg (n=4; P<0.02), 28.7±5 mm Hg
(n=4; P<0.02), and 25.7±5.6 mm Hg (n=4;
P<0.02) after 20, 30, and 40 minutes after administration,
respectively (Figure 3C).
Biotin–SKGR-chloromethylketone (0.1 mg/kg IV), a
potent irreversible inhibitor of trypsin
proteolysis,13 given 30 minutes before trypsin (2.4
mg/kg) reduced the hypotensive response significantly, from 41.5±6.4
to 21±6.9 mm Hg (n=5; P<0.01). Strikingly, none of
the drugs tested, including the trypsin inhibitor, caused a
significant inhibition of the PAR-2AP–induced hypotension. Indeed, at
a dose of 0.3 mg/kg IV, PAR-2AP gave a hypotension of 27.3±6.3
mm Hg (n=6) that was not significantly inhibited by
indomethacin 5 mg/kg (27±6.3 mm Hg; n=5;
P=NS), L-NAME 3 µg ·
kg-1 · min-1
(28±3 mm Hg; n=4; P=NS), SB209670 1 mg/kg
(27±2.7 mm Hg; n=4; P=NS),
biotin–SKGR-chloromethylketone 0.1 mg/kg
(26.5±4.8 mm Hg, n=4, P=NS), or Hirulog 10 mg/kg
(20±5 mm Hg; n=3; P=NS), a specific thrombin
inhibitor.
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Immunolocalization and RT-PCR Analysis of PAR-2 in
LPS-Treated Rats
Polyclonal IgGs to PAR-2 were produced in rabbits by use of a
multiple antigenic peptide14 corresponding to the
activation site of human PAR-2
(SKGRSLIGKVDGTSHVTGK-NH2, residues 33 to 51). The
resulting antibodies were purified from rabbit serum with an affinity
chromatography column consisting of linear peptide
SKGRSLIGKVDGTSHVTGK-NH2 (peptide was synthesized
as a C-terminal amide) coupled to Sepharose as reported
previously.15 The specificity of the PAR-2 antibodies was
established in flow cytometric and immunohistochemical
studies.9 10 Because LPS had been shown to upregulate
PAR-2 expression in endothelial cells in vitro, we
investigated expression of PAR-2 in blood vessels obtained from normal
and LPS-treated rats. Control and PAR-2 antibodies at a range of
dilutions (1, 5, 10, and 20 µg/mL) were used for
immunohistochemistry, and the staining intensities obtained with PAR-2
and control antibodies were compared at the same concentration. PAR-2
staining was apparent in endothelial cells and vascular
smooth muscle cells within the blood vessels obtained from normal rats
and rats treated with LPS 20 hours before death.
Endothelial cells were identified in sequential
sections by staining for factor VIII antigen (Figure 4A).
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The expression of PAR-2 after LPS administration in vivo was
investigated by semiquantitative RT-PCR analysis (Figure 4B
). Primers based on the rat PAR-2 sequence were used with cDNA
of total RNA obtained from the aortas and jugular veins of rats treated
with LPS or saline 20 hours before death. The increased intensity of
the PAR-2 PCR product bands (1900 bp) obtained (Figure 4)
indicated that expression of PAR-2 mRNA was increased in both the aorta
and jugular veins of LPS-treated rats compared with saline-treated
controls.
Effect of PAR-2AP and Trypsin on LPS-Treated Rats
PAR-2AP– and trypsin-induced hypotensive responses were
significantly increased in those rats given LPS 20 hours earlier.
Initial MABP in LPS-treated rats was 127.4±4.12 mm Hg. However, if
experiments were performed 6 or 12 hours after LPS administration,
there was no change in hypotension induced by either PAR-2AP or
trypsin. As shown in Figure 5A, in
LPS-treated rats (13.5x106 U/kg IV 20 hours
earlier), the hypotension caused by PAR-2AP (0.1 mg/kg IV) was
21.7±1.87 mm Hg, versus 6.2±1.59 mm Hg in saline-treated rats
(n=4; P<0.001). PAR-2AP given at 0.03 mg/kg did not cause
hypotension in normal rats, but strikingly, the hypotension in
LPS-treated rats was 20.7±3.2 mm Hg (n=4; P<0.001;
Figure 5A). The hypotensive response to higher doses (0.3 and 1
mg/kg) was not affected significantly by LPS (data not shown). As shown
in Figure 5B, the potency of trypsin in the induced hypotensive
response was also significantly increased in LPS-treated rats compared
with normal rats. At the doses of 0.2 and 0.6 mg/kg, trypsin
hypotension was 26±3.64 and 36.2±1.99 mm Hg in LPS-treated rats
versus 9.87±1.03 and 23.9±1.55 mm Hg in controls (n=4;
P<0.001); a lower dose (0.02 mg/kg) was inactive. All the
inhibitors tested had no effect on the hypotension induced
by trypsin (0.2 to 0.6 mg/kg) or PAR-2AP (0.3 and 0.1 mg/kg). In
separate experiments, the hypotensive response in LPS-treated rats to
GTN (0.5 mg/kg IV) and bradykinin (10 µg/kg IV) was also evaluated.
Hypotension produced by GTN in normal rats (Figure 5C) was not
significantly different from that produced in LPS-treated rats
(26±2.93 versus 27.1±2.43 mm Hg). Conversely, bradykinin-induced
hypotension was enhanced significantly, from 36.3±1.29 mm Hg in
normal rats to 50±2.29 mm Hg (P<0.001) in
LPS-treated rats (Figure 5D).
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The 10-fold reduction in the minimum effective dose of agonist peptide
to induce hypotension in LPS-treated rats was striking. As shown in
Figure 6, both the kinetics and magnitude
of the hypotension induced by PAR-2AP in LPS-treated rats were
identical to those seen in control animals at a 10-fold higher
concentration of peptide. These data suggest a functional upregulation
of PAR-2 in vivo.
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In Vitro Studies
Aortic rings from normal rats precontracted with PE relaxed in a
concentration-dependent manner to PAR-2AP (10, 30, and 100
µmol/L) and trypsin. Both responses were significantly potentiated in
aortic rings from LPS-treated rats and abolished by L-NAME 100
µmol/L (Figure 7). Removal of
endothelium caused a complete loss of the relaxing
effect of both trypsin and PAR-2AP.
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) or LPS-treated (
,
13.5x106 U/kg IV) rats 20 hours before experiment. B,
Effect of trypsin-induced relaxation in aorta rings collected from
vehicle-treated (
) or LPS-treated (
) rats.
Because L-NAME completely abolished effect, fourth curve is perfectly
overlapping and does not appear in graph. Results are expressed as
mean±SEM of 4 experiments. Percent of relaxation induced by PAR-2AP is
calculated as percent of maximum relaxation obtained with acetylcholine
in same tissue. |
Discussion |
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Intravenous administration of trypsin was followed almost immediately by a dose-related hypotension. When PAR-2AP was tested under the same experimental conditions, it caused a hypotensive response that was very similar. Our results are consistent with those obtained previously by others, showing that in vivo activation of PAR-2 causes hypotension.16 17 18 On the basis of the earliest studies of Rocha e Silva,1 2 3 we evaluated the involvement of histamine in the trypsin effect on blood pressure. The H1 antagonist mepyramine had no effect on trypsin or PAR-2AP–induced hypotension. These data are in agreement with an earlier study that demonstrated that trypsin-induced hypotension in vivo is resistant to antithistaminic drugs.4 We investigated the possible mediators involved in the hypotensive response induced by trypsin or PAR-2AP, using a set of inhibitors as pharmacological tools. For this purpose, we used atropine; a cyclooxygenase inhibitor, indomethacin; a bradykinin B2 antagonist, HOE140; an ETA/ETB endothelin antagonist, SB-209670X; and a nitric oxide inhibitor, L-NAME. Trypsin hypotension was inhibited by indomethacin, SB-209670X ETA/ETB, and L-NAME. We also used a trypsin inhibitor, biotin–SKGR-chloromethylketone, a peptide that specifically blocks the proteolytic activity of trypsin.13 The finding that trypsin-induced hypotension was inhibited by biotin–SKGR-chloromethylketone indicates that proteolysis is required for the induction of hypotension. Unexpectedly, and strikingly, none of these inhibitors affected the hypotension caused by PAR-2AP. Similarly, in vitro experiments have shown that PAR-2AP–induced relaxation of rat vascular tissues is not affected by a broad range of inhibitors, including atropine, indomethacin, chlorpheniramine, genistein, propranolol, or ritanserin,19 but only by nitric oxide synthase inhibition.16 19 20 When we tested the effect of PAR-2AP in vitro on precontracted rat aortic rings, the relaxant effect was clearly endothelium-dependent and blocked by L-NAME, confirming the in vitro data obtained previously.16 19 20 Furthermore, we observed a potentiation of relaxation of aortic rings from LPS-treated rats similar to that observed in vivo. The discrepancy between the effects of L-NAME on PAR-2AP actions in vitro and in vivo most likely reflects other interactions that contribute to the hypotension in vivo, whereas in vitro, the effect is completely dependent on nitric oxide. It also seems likely that trypsin activates multiple signaling pathways in vivo, whereas the agonist peptide–induced PAR-2 activation involves specific pathways, unaffected by the inhibitors, which remain to be identified.
To exclude the possibility that thrombin generated after the administration of trypsin may be mediating the hypotensive response, we treated rats with Hirulog, a specific inhibitor of thrombin, before administration of trypsin and PAR-2AP. Neither the PAR-2AP nor trypsin responses were inhibited in rats pretreated with Hirulog. However, we cannot exclude a role for the thrombin receptor, because high concentrations of trypsin can cleave and activate PAR-1 as well as PAR-2. Importantly, by contrast, the PAR-2–activating peptide PAR-2AP is relatively specific and does not activate PAR-1.21 Therefore, the differences between the responses to trypsin compared with those induced by PAR-2AP in vivo may be due, in part, to the simultaneous activation of PAR-1 and PAR-2 by trypsin but of PAR-2 alone by PAR-2AP. This raises interesting questions about what effects PAR-1 and PAR-2 activation together may have on blood pressure. Some authors have suggested that PAR-1 activation produces a biphasic change in blood pressure, whereas PAR-2 activation alone may cause a single-phase hypotensive response,16 18 and our data would support the latter.
After stimulation of endothelial cells with LPS, PAR-2 mRNA is elevated 5- to 10-fold, whereas the thrombin PAR-1 receptor expression is unaffected.9 Therefore, we extended this study to test the possible role of PAR-2 in the hypotension of endotoxemic rats and to evaluate whether endotoxin has a role in the regulation of PAR-2 receptor in vivo. Because hypotension is one of the main features of septic shock, we evaluated in vivo whether the hypotensive response to PAR-2AP and trypsin was modified by LPS and whether expression of PAR-2 was concomitantly increased in artery and vein ex vivo. PAR-2 was immunolocalized to smooth muscle and endothelial cells in aorta and jugular vein, which supports reports showing PAR-2 expression in these cells.9 10 19 20 Clearly increased expression of PAR-2 in both the aorta and jugular veins in LPS-treated rats also was shown by RT-PCR. Moreover, the LPS-treated rats were more sensitive to the effects of both PAR-2AP and trypsin compared with normal rats, consistent with functional upregulation of PAR-2 by LPS in vivo. Indeed, after LPS treatment, the hypotension was induced with a concentration of PAR-2AP (0.03 mg/kg IV) 10-fold lower than that used in normal controls to give the same effect (0.3 mg/kg IV). Similarly, the hypotensive response to trypsin was increased significantly at both 0.2 and 0.6 mg/kg. These findings raise the significant possibility that PAR-2 may play a important role in the hypotension associated with endotoxemia and septic shock. Interestingly, a drug called ulinastatin,22 23 a trypsin inhibitor produced by the liver in response to a range of stimuli, including infection, is currently used in Japan to treat septic shock. However, a key question that remains to be resolved is the identity of relevant pathophysiological protease ligand for PAR-2, because trypsin is not found in the circulation, except perhaps during gastrointestinal tract surgery.
In conclusion, we have shown that trypsin and a PAR-2AP cause a dose-dependent hypotension in normal rats and that the hypotensive effect was exacerbated by pretreating rats with LPS. PAR-2AP caused a sustained hypotension at a concentration as low as 30 ng/kg, well within the concentration range used in vitro to assess the effects of LPS on PAR-2AP responses in endothelial cells. Increased vascular expression of PAR-2, immunolocalized to endothelial and smooth muscle cells, was also demonstrated in LPS-treated rats by RT-PCR, and a marked functional upregulation of the receptor was shown by a 10-fold lower dose of PAR-2AP inducing hypotension in LPS-treated rats. These data strongly indicate a potentially important role for PAR-2 hypotension and endotoxemia. Unraveling the roles of PAR-2 in endotoxemia, along with those of PAR-1 and the newly cloned PAR-3, may lead to the development of new therapeutic strategies and targets.
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Acknowledgments |
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This work was supported in part by grants from the Wellcome Trust, Special Trustees of the Royal London Hospital, and by Ministero della Ricerca Scientifica e Tecnologica (MURST).
Received August 11, 1998; revision received February 16, 1999; accepted February 16, 1999.
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